Human monoclonal antibodies against interleukin 8 (IL-8)

ABSTRACT

Isolated human monoclonal antibodies which bind to IL-8 (e.g., human IL-8) are disclosed. The human antibodies can be produced in a hybridoma, transfectoma or in a non-human transgenic animal, e.g., a transgenic mouse, capable of producing multiple isotypes of human monoclonal antibodies by undergoing V-D-J recombination and isotype switching. Also disclosed are pharmaceutical compositions comprising the human antibodies, non-human transgenic animals, hybridomas, and transfectomas which produce the human antibodies, and therapeutic and diagnostic methods for using the human antibodies.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/074,226, filed on Nov. 7, 2013, which is a divisional of U.S. patentapplication Ser. No. 13/337,798, filed on Dec. 27, 2011, which is adivisional of U.S. patent application Ser. No. 12/616,615, filed on Nov.11, 2009, which is a continuation of U.S. patent application Ser. No.11/823,481, filed on Jun. 27, 2007, which is a continuation of U.S.patent application Ser. No. 10/738,120, filed Dec. 16, 2003, whichclaims the benefit of U.S. Provisional Patent Application Ser. No.60/433,728, filed Dec. 16, 2002, the entire contents of which areincorporated herein by this reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 15, 2016, isnamed GMI-278CN2DV2CN_Sequence Listing.txt and is 18,507 bytes in size.

BACKGROUND OF THE INVENTION

Chemokines represent a superfamily of about 30 chemotactic cytokinesacting as vital initiators and promulgators of inflammatory reactions.They range from 8 to 11 kD in molecular weight, are active over a 1 to100 ng/mL concentration range, and are produced by a wide variety ofcell types.

Interleukin 8 (IL-8), formerly called monocyte-derived neutrophilchemotactic factor (MDNCF) or neutrophil attractant/activation protein-1(NAP-1), is a chemokine and a member of the cytokine family thatdisplays chemotactic activity for specific types of leukocytes. IL-8 isa member of the CXC chemokine family in which an amino acid is presentbetween the first two of four highly conserved cysteine residues. IL-8is a polypeptide of which two predominant forms consist of 72 aminoacids and 77 amino acids. Monocytes, macrophages, neutrophils,lymphocytes, dermal fibroblasts, keratinocytes, vascular endothelialcells, melanocytes, hepatocytes, and various tumor cell lines produceIL-8. IL-8 is a potent neutrophil chemokine and participates in themigration of neutrophils towards inflammatory sites. Upon binding to itshigh-affinity receptors (CXCR1 and CXCR2) which are present on thesurface of neutrophils, IL-8 activates neutrophils by acceleratingdegranulation and elevating the free Ca²⁺ concentration in the cytoplasmand also induces neutrophil migration to thereby destroy the infiltratedtissue.

Although the neutrophil inflammatory response is essential for thedestruction of bacteria which are invading the body, inappropriateneutrophil activation can cause a number of inflammatory disorders. Forexample, IL-8 has been recovered from inflammatory sites such as,pustulosis palmoplantaris (PPP) lesions, psoriatic scales, synovialfluid of patients with rheumatoid arthritis (RA), pleural fluid fromempyema patients, alveolar macrophages from lungs with idiopathicpulmonary fibrosis, broncheoalveolar lavage fluids from patients withadult respiratory distress syndrome, cystic fibrosis, chronicbronchitis, and bronchiectasis. IL-8 is also associated with sepsis,asthma, glomerulonephritis, inflammatory bowel disease (IBD),ischaemia-reperfusion injury and multiple myeloma. Such conditions arecharacterized by inflammation accompanied by neutrophil infiltration andtissue damage.

IL-8 is also known to promote angiogenesis and, thus, growth of tumors.Such activity has been associated with the ELR motif within the IL-8sequence. Human tumor cell lines such as, thyroid carcinoma,transitional cell carcinoma, trichilemmona, squamous cell carcinoma, andmelanoma constitutively express IL-8 which plays a role in tumorinvasion and metastasis.

Accordingly, antibodies specific for IL-8 are therapeutically importantfor treating diseases mediated by IL-8 activity. A hybridoma producing ahuman antibody against human IL-8, referred to as 2C6, has beendescribed previously (U.S. Pat. No. 6,300,129 by Lonberg and Kay).However, additional antibodies specific for IL-8 are still needed.

SUMMARY OF THE INVENTION

The present invention provides isolated human monoclonal antibodieswhich bind to human IL-8, as well as bispecific and multispecificmolecules and other therapeutic compositions containing such antibodies,alone or in combination with additional therapeutic agents. Alsoprovided are methods for treating a variety IL-8 mediated diseases usingthe antibodies and compositions of the invention.

The fully human antibodies of the present invention bind to IL-8 andinhibit IL-8 function (and IL-8 mediated effects) by blocking IL-8binding to its receptor. For example, the antibodies can inhibitproinflammatory and angiogenic effects induced by IL-8, such as IL-8induced chemotactic activity for leukocytes and IL-8 induced calciumflux. The antibodies can also inhibit IL-8 induced increased expressionof CD11b (Mac-1) and decreased expression of L-selectin (CD62L).Accordingly, particular antibodies of the invention have one or more ofthe following characteristics:

(i) inhibits IL-8 binding to its receptors (CXCR1 and CXCR2);

(ii) inhibits IL-8 induced proinflammatory effects;

(iii) inhibits IL-8 induced chemotactic activity for neutrophils;

(iv) inhibits IL-8 induced calcium flux;

(v) inhibits IL-8 induced changes in expression levels of adhesionmolecules on neutrophils;

(vi) inhibits IL-8 induced increased expression of CD11b (Mac-1) andinhibits IL-8 induced decreased expression of L-selectin on neutrophils;

(vii) does not cross-react with related chemokines selected from thegroup consisting of human GRO-α, human GRO-β, human IP-10 and humanNAP-2;

(viii) significantly inhibits chemotaxis induced by biological fluidswhich contain multiple chemotactic factors including IL-8.

Therefore, the human antibodies of the present invention provide animproved means for treating and preventing disorders mediated by IL-8activity attributable in part to their unique specificity (e.g., epitopespecificity and lack of cross-reactivity with related chemokines),affinity, structure, functional activity and the fact that they arefully human, making them significantly less immunogenic and moretherapeutically effective and useful when administered to human patientsthan other IL-8 antibodies previously generated (e.g., murine andhumanized antibodies).

Isolated human antibodies of the invention include a variety of antibodyisotypes, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, secretoryIgA, IgD, and IgE. Typically, they include IgG1 (e.g., IgG1,κ orIgG1,λ), IgG3, IgG4 and IgM isotypes. The antibodies can be intact(e.g., an IgG1 or IgG3 antibody) or can include only an antigen-bindingportion (e.g., a Fab, F(ab′)₂, Fv or a single chain Fv fragment).

Particular therapeutic antibodies of the invention include humanmonoclonal antibody (HuMab) 10F8 and functionally equivalent antibodieswhich (a) are encoded by human heavy chain and human light chain nucleicacids comprising nucleotide sequences in their variable regions as setforth in SEQ ID NO:10 and SEQ ID NO:6, respectively, and sequences whichare at least 95% homologous therewith, or (b) include heavy chain andlight chain variable regions which comprise the amino acid sequencesshown in SEQ ID NO:12 and SEQ ID NO:8, respectively, and sequences whichare at least 95% homologous therewith.

Still other particular human antibodies of the invention include thosewhich comprise a CDR domain having a human heavy and light chain CDR1region, a human heavy and light chain CDR2 region, and a human heavy andlight chain CDR3 region, wherein the antibody comprises at least one CDRsequence selected from the group consisting of: V_(L) CDR1 of SEQ ID NO:16, V_(L) CDR2 of SEQ ID NO: 17, V_(L) CDR3 of SEQ ID NO: 18, V_(H) CDR1of SEQ ID NO: 22, V_(H) CDR2 of SEQ ID NO: 23, and V_(H) of SEQ ID NO:24. Antibodies which comprise at least the V_(H) CDR3 of SEQ ID NO: 24are also encompassed by the present invention, as well as antibodieswhich comprise at least four CDR sequences selected from the groupconsisting of: V_(L) CDR1 of SEQ ID NO: 16, V_(L) CDR2 of SEQ ID NO: 17,V_(L) CDR3 of SEQ ID NO: 18, V_(H) CDR1 of SEQ ID NO: 22, V_(H) CDR2 ofSEQ ID NO: 23, and V_(H) of SEQ ID NO: 24 and antibodies which comprisethe six CDR sequences: V_(L) CDR1 of SEQ ID NO: 16, V_(L) CDR2 of SEQ IDNO: 17, V_(L) CDR3 of SEQ ID NO: 18, V_(H) CDR1 of SEQ ID NO: 22, V_(H)CDR2 of SEQ ID NO: 23, and V_(H) of SEQ ID NO: 24.

The present invention further includes antibodies which bind to anepitope on human IL-8 defined by antibody 10F8, and/or which compete forbinding to IL-8 with antibody 10F8, or which have other functionalbinding characteristic exhibited by antibody 10F8. Such antibodiesinclude those which bind to IL-8 with a dissociation equilibriumconstant (K_(D)) of approximately 10⁻⁸M or less, 10⁻⁹ M or less, 10⁻¹⁰ Mor less, or 10⁻¹¹ M or even less. Such antibodies also include thosewhich do not cross-react with related chemokines, e.g., human GRO-α,human GRO-β, IP-10 or human NAP-2, and thus do not inhibit theirfunction.

In another aspect, human antibodies of the invention can beco-administered with one or more further therapeutic agents. They can becoadministered simultaneously with such agents (e.g., in a singlecomposition or separately) or can be administered before or afteradministration of such agents. Such further agents can include agentsfor treating inflammatory or hyperproliferative skin disorders,immunosuppressive agents, anti-inflammatory agents, or chemotherapeuticagents.

In another aspect, the present invention provides compositions, e.g.,pharmaceutical or diagnostic compositions, comprising one or more (i.e.,a combination of) human anti-IL-8 antibodies together with apharmaceutically acceptable carrier. The composition can further includeone or more other therapeutic agents, such as those disclosed above.

For use in in vivo treatment and prevention of IL-8 mediated diseases,human antibodies of the present invention are administered to patients(e.g., human subjects) at therapeutically effective dosages using anysuitable route of administration, such as injection or infusion andother routes of administration known in the art for antibody-basedclinical products.

In yet another aspect, the invention provides methods for inhibiting theproinflammatory effects of IL-8, such as IL-8 induced chemotacticactivity for leukocytes.

Accordingly, human antibodies of the present invention can be used totreat and/or prevent a variety of IL-8 mediated diseases byadministering the antibodies to patients suffering from such diseases.

Exemplary diseases that can be treated (e.g., ameliorated) or preventedusing the methods and compositions of the invention include, but are notlimited to, inflammatory or hyperproliferative skin disorders, immune,autoimmune, inflammatory or infectious diseases, and diseases involvingIL-8 mediated angiogenesis, such as tumors and cancers.

In yet another aspect, the present invention provides a method fordetecting in vitro or in vivo the presence of IL-8 in a sample or anindividual, e.g., for diagnosing an IL-8-related disease. This can alsobe useful for monitoring a IL-8 related disease and the effect oftreatment with an anti-IL-8 antibody and for determining and adjustingthe dose of the antibody to be administered. In one embodiment, thepresence of IL-8 is detected by contacting a sample to be tested,optionally along with a control sample, with a human monoclonal antibodyof the invention under conditions that allow for formation of a complexbetween the antibody and IL-8. Complex formation is then detected (e.g.,using an ELISA). When using a control sample along with the test sample,complex is detected in both samples and any statistically significantdifference in the formation of complexes between the samples isindicative of the presence of IL-8 in the test sample.

In a further aspect, the invention relates to anti-idiotypic antibodieswhich bind to the human monoclonal antibodies of the invention. Theseanti-idiotypic antibodies can be used as an immunodiagnostic tool todetect and quantify levels of human monoclonal antibodies against IL-8in laboratory or patient samples. This may be useful for examiningpharmacokinetics of the anti-IL-8 antibody or for determining andadjusting the dosage of the anti-IL-8 antibody and for monitoring thedisease and the effect of treatment in a patient.

Mouse anti-idiotypic antibodies can be made, e.g., by immunizing BALB/Cmice with the human monoclonal antibodies according to the invention,and generating hybridomas from spleens of these mice by fusion withmyeloma cells, such as NS1 cells, using standard techniques.

In yet another aspect, the invention provides a transgenic non-humananimal, such as a transgenic mouse, which express human monoclonalantibodies that bind to IL-8. In a particular embodiment, the transgenicnon-human animal is a transgenic mouse having a genome comprising ahuman heavy chain transgene and a human light chain transgene encodingall or a portion of an antibody of the invention. The transgenicnon-human animal can be immunized with a purified or enrichedpreparation of IL-8 antigen, recombinant IL-8 antigen and/or cellsexpressing IL-8, including cells transfected with IL-8. Preferably, thetransgenic non-human animal, e.g., the transgenic mouse, is capable ofproducing multiple isotypes of human monoclonal antibodies to IL-8(e.g., IgG, IgA and/or IgM) by undergoing V-D-J recombination andisotype switching. Isotype switching may occur by, e.g., classical ornon-classical isotype switching.

Accordingly, in yet another aspect, the invention provides isolated Bcells from a transgenic non-human animal as described above, e.g., atransgenic mouse, which expresses human anti-IL-8 antibodies. Theisolated B cells can then be immortalized by fusion to an immortalizedcell to provide a source (e.g., a hybridoma) of human anti-IL-8antibodies. Such hybridomas (i.e., which produce human anti-IL-8antibodies) are also included within the scope of the invention.

As exemplified herein, human antibodies of the invention can be obtaineddirectly from hybridomas which express the antibody, or can be clonedand recombinantly expressed in a fju′\m (e.g., a CHO cell, or a NS/0cell). Further examples of host cells are HEK293 cells, plant cells,microorganisms, such as E. coli, and fungi, such as yeast.Alternatively, they can be produced recombinantly in a transgenicnon-human animal or plant.

Accordingly, in yet another aspect, the invention provides nucleic acidmolecules encoding human anti-IL-8 antibodies, as well as recombinantexpression vectors which include the nucleic acids of the invention, andhost cells transfected with such vectors. Methods of producing theantibodies by culturing these host cells are also encompassed by theinvention. Particular nucleic acids provided by the invention comprisethe nucleotide sequences shown in SEQ ID NO:10 and SEQ ID NO:6, encodingto the heavy and light chains respectively of 10F8.

Other features and advantages of the instant invention will be apparentfrom the following detailed description and examples which should not beconstrued as limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequences of the V_(L)- and V_(H)-regions(SEQ ID NOs:1 and 3), respectively, from HuMab 10F8. Leader sequencesare underlined.

FIG. 2 is an alignment comparison of the nucleotide sequence of thelight (kappa) chain V region of HuMab 10F8 (SEQ ID NO:6) and thecorresponding Vκ A-27 germline nucleotide sequence (SEQ ID NO:5).

FIG. 3 is an alignment comparison of the amino acid sequence of thelight (kappa) chain V region of HuMab 10F8 (SEQ ID NO:8) and thecorresponding Vκ A-27 germline-encoded amino acid sequence (SEQ IDNO:7).

FIG. 4 is an alignment comparison of the nucleotide sequence of theheavy chain V region of HuMab 10F8 (SEQ ID NO:10) and the correspondingV_(H) 3-33 germline nucleotide sequence (SEQ ID NO:9).

FIG. 5 is an alignment comparison of the amino acid sequence of theheavy chain V region of HuMab 10F8 (SEQ ID NO:12) and the correspondingV_(H) 3-33 germline-encoded amino acid sequence (SEQ ID NO:11).

FIG. 6 is a graph showing that HuMab 10F8 binds to both endothelial cellderived and monocyte derived IL-8, but that it does not bind to IP-10,GRO-α or GRO-β.

FIG. 7A is a graph showing inhibition of [¹²⁵I]-IL-8 binding toneutrophils by HuMab 10F8 (open squares) as compared to a murineIL-8-specific antibody (m-a-IL8) (asterisks).

FIG. 7B is a graph showing inhibition of [¹²⁵I]-M-8 binding toneutrophils by hybridoma-derived HuMab 10F8 (10F8 H) (open squares) andtransfectoma-derived HuMab 10F8 (10F8 T) (closed squares), respectively.

FIG. 8A is a graph showing inhibition of IL-8 mediated neutrophilchemotaxis by HuMab 10F8 (triangles) as compared to a murineIL-8-specific antibody (6217.111) (squares).

FIG. 8B is a graph showing inhibition of IL-8 mediated neutrophilchemotaxis by HuMab 10F8 as determined by a transmigration assay using aBoyden chamber.

FIG. 9A is a graph showing inhibition of IL-8 mediated shedding ofL-selectin (CD62L) on the surface of neutrophils by HuMab 10F8 (closedsquares) as compared to an irrelevant human isotype control antibody(asterisks).

FIG. 9B is a graph showing inhibition of IL-8 mediated expression ofCD11b on the cell surface of neutrophils by HuMab 10F8 (closed squares)as compared to an irrelevant human isotype control antibody (asterisks).

FIG. 10 is a graph showing the presence of IL-8 and GRO-α in pustulosispalmoplantaris (PPP) patient material as determined by ELISAs.

FIG. 11 shows the results of the measurement of IL-8, GRO-α and C5apresent in feet water fluid obtained from healthy controls (n=6), eczemapatients (n=6) or PPP patients (n=6).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved human antibodies which bind tohuman IL-8 and antibody-based therapies for treating and diagnosing avariety of disorders mediated by IL-8 (e.g., disorders caused by theproinflammatory effects and the angiogenic effects of IL-8). As usedherein, the term “proinflammatory effects” includes any humoral orcell-mediated immune response induced by IL-8, such as the chemotacticactivity for leukocytes. The term “angiogenic effects of IL-8” includesthe growth of new blood vessels or the vascularization of tumor cellsinduced by IL-8. Therapies of the invention employ human monoclonalantibodies which bind to and inhibit such functions of IL-8,particularly in human therapy.

In one embodiment the antibody is an IgG1 antibody, more particularly anIgG1,κ or IgG1,λ, isotype. In another embodiment the antibody is an IgG3antibody, more particularly an IgG3,κ or IgG3,λ isotype. In yet anotherembodiment the antibody is an IgG4 antibody, more particularly an IgG4,κor IgG4,λ isotype. In still another embodiment the antibody is an IgA1or IgA2 antibody. In yet a further embodiment the antibody is an IgMantibody.

In one embodiment, the human antibodies are produced in a non-humantransgenic animal, e.g., a transgenic mouse, capable of producingmultiple isotypes of human monoclonal antibodies to IL-8 (e.g., IgG, IgAand/or IgE) by undergoing V-D-J recombination and isotype switching.Such transgenic animal can also be a transgenic rabbit for producingpolyclonal antibodies such as disclosed in US 2003/0017534. Accordingly,the invention also encompasses human polyclonal antibodies whichspecifically bind to IL-8. Accordingly, particular aspects of theinvention include not only antibodies, antibody fragments, andpharmaceutical compositions thereof, but also non-human transgenicanimals, B cells, hybridomas, and transfectomas which produce monoclonalantibodies. Methods of using the antibodies of the invention to detect acell producing IL-8, either in vitro or in vivo, are also encompassed bythe invention. Methods of using the antibodies of the invention to blockor inhibit IL-8 induced activities, e.g., proinflammatory activities,chemotactic activities, and angiogenesis are also provided and areuseful in the treatment of disorders associated with IL-8.

In one embodiment, the human antibodies of the invention can be used inmethods for treating inflammatory or hyperproliferative skin disorders,such as pustulosis palmoplantaris (PPP), psoriasis, including plaquepsoriasis and guttate type psoriasis, bullous skin diseases, such asbullous pemphigoid, contact dermatitis, eczema, erythematosus, andatopic dermatitis.

In another embodiment, the human antibodies of the invention can be usedin methods for treating immune, autoimmune, inflammatory or infectiousdiseases, such as psoriatic arthritis, systemic scleroderma andsclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerativecolitis, acute lung injury, such as acute respiratory distress syndromeor adult respiratory distress syndrome, meningitis, encephalitis,uveitis, multiple myeloma, glomerulonephritis, nephritis, asthma,atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis,Raynaud's syndrome, Sjögren's syndrome, juvenile onset diabetes,Reiter's disease, Behcet's disease, immune complex nephritis, IgAnephropathy, IgM polyneuropathies, immune-mediated thrombocytopenias,such as acute idiopathic thrombocytopenic purpura and chronic idiopathicthrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupusnephritis, lupus erythematosus, rheumatoid arthritis (RA), ankylosingspodylitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, smallvessel vasculitides, such as Wegener's granulomatosis, Omen's syndrome,chronic renal failure, autoimmune thyroid disease, acute infectiousmononucleosis, HIV, herpes virus associated diseases, human virusinfections, such as common cold as caused by human rhinovirus,coronavirus, other enterovirus, herpes virus, influenza virus,parainfluenza virus, respiratory syncytial virus or adenovirusinfection, bacteria pneumonia, wounds, sepsis, cerebral stroke/cerebraledema, ischaemia-reperfusion injury and hepatitis C.

In one embodiment, the human monoclonal antibodies can be used for thetreatment of ischaemia-reperfusion injury after thrombolysis,cardiopulmonary bypass, percutaneous coronary intervention (PCI),coronary artery bypass, or cardiac transplantation.

In yet another embodiment, the human antibodies of the invention can beused for treatment of alcoholic hepatitis and acute pancreatitis.

In yet a further embodiment, the human antibodies of the invention canbe used in methods for treating diseases involving IL-8 mediatedangiogenesis, such as tumors and cancers, e.g., melanoma, thyroidcarcinoma, transitional cell carcinoma, trichilemmona, squamous cellcarcinoma and breast cancer.

In another embodiment, the human antibodies of the invention can be usedfor treating diseases wherein blocking of granulocyte migration isbeneficial, e.g., in

diseases affecting the central nervous system, such as isolated cerebralangiitis;

diseases affecting the peripheral nervous system, such as mononeuritismultiplex;

cardiovascular disorders, such as acute myocardial infarction,myocarditis, pericarditis, and Liebman-Sachs endocarditis;

pulmonary disorders, such as chronic obstructive pulmonary disease(COPD), alveolitis, obliterating bronchiolitis, cystic fibrosis,allergic aspergillosis, and Löfflers syndrome;

hepatic disorders, such as sclerosing cholangiolitis;

urogenital disorders, such as chronic cyctitis;

renal disorders, such as tubulo-interstial nephritis;

infectious diseases, such as severe acute respiratory syndrome (SARS);

rheumatic disorders, such as large vessel vasculitides (including giantcell arteritis, polymyalgia rheumatica, and Takayasu arteritis),medium-sized vessel vasculitides (including polyarteritis nodosa,localized polyarteritis nodosa, and Kawasaki disease), small vesselvasculitides (including Churg-Strauss syndrome, microscopicpolyarteritis, cryoglobulinemic vasculitis, and leucocytoclasticangiitis), secondary vasculitides (including rheumatoid vasculitis, andvasculitis associated with systemic lupus erythematosus or Sjögren'ssyndrome), isolated sacroileitis, the SAPHO syndrome, and disciitis(including postoperative disciitis);

endocrine disorders, such as subacute thyroiditis;

skin disorders, such as cicatricial pemphigoid, dermatitisherpetiformis, subcorneal pustular dermatosis, epidermolysis bullosaacquisita, rosacea, acute febrile dermatosis, granuloma annulare(including Sweet's syndrome), pyoderma gangraenosum, and acne (includingacne conglobata);

connective tissue disorders, such as sarcoidosis, relapsingpolychondritis, familial Mediterranean fever, panniculitis, erythemanodosum, Weber-Christian's disease, and retroperitoneal fibrosis.

In another embodiment, the human antibodies of the invention are usedfor treating diseases wherein interfering with interactions between IL-8and osteoclasts is beneficial, such as osteoporosis, and osteolyticmetastases.

In another embodiment, the human antibodies of the invention are usedfor treating disease wherein interfering with interactions between IL-8and tumor cells is beneficial, such as gastric cancer, colorectalcancer, and urine bladder cancer.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The terms “IL-8,” “IL-8 antigen” and “Interleukin 8” are usedinterchangeably herein, and include any variants or isoforms which arenaturally expressed by cells or are expressed by cells transfected withthe IL-8 gene.

The term “antibody” as referred to herein includes intact antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asV_(H)) and a heavy chain constant region. Each light chain is comprisedof a light chain variable region (abbreviated herein as V_(L)) and alight chain constant region. The V_(H) and V_(L) regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each V_(H) and V_(L) iscomposed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4. The variable regions of the heavy and light chains contain abinding domain that interacts with an antigen. The constant regions ofthe antibodies may mediate the binding of the immunoglobulin to hosttissues or factors, including various cells of the immune system (e.g.,effector cells) and the first component (C1q) of the classicalcomplement system.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to bind to an antigen (e.g., IL-8). Ithas been shown that the antigen-binding function of an antibody can beperformed by fragments of an intact antibody. Examples of bindingfragments encompassed within the term “antigen-binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe V_(L), V_(H), C_(L) and C_(H1) domains; (ii) a F(ab′)₂ fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the V_(H)and C_(H1) domains; (iv) a Fv fragment consisting of the V_(L) and V_(H)domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,(1989) Nature 341:544-546), which consists of a V_(H) domain; and (vi)an isolated complementarity determining region (CDR), e.g., V_(H) CDR3.Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv); see e.g.,Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies arealso intended to be encompassed within the term “antigen-bindingportion” of an antibody. Furthermore, the antigen-binding fragmentsinclude binding-domain immunoglobulin fusion proteins comprising (i) abinding domain polypeptide (such as a heavy chain variable region, alight chain variable region, or a heavy chain variable region fused to alight chain variable region via a linker peptide) that is fused to animmunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavychain CH2 constant region fused to the hinge region, and (iii) animmunoglobulin heavy chain CH3 constant region fused to the CH2 constantregion. The hinge region is preferably modified by replacing one or morecysteine residues with serine residues so as to prevent dimerization.Such binding-domain immunoglobulin fusion proteins are further disclosedin US 2003/0118592 and US 2003/0133939. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost by treatment with denaturing solvents.

As used herein, the terms “inhibits binding” and “blocks binding” (e.g.,referring to inhibition/blocking of binding of IL-8 to its receptors,CXCR1 and CXCR2) are used interchangeably and encompass both partial andcomplete inhibition/blocking. The inhibition/blocking of IL-8 preferablyreduces or alters the normal level or type of activity that occurs whenIL-8 binding occurs without inhibition or blocking, e.g., inhibition ofIL-8 induced elastase release or calcium flux or inhibition of IL-8induced increased expression of CD11b (Mac-1) and decreased expressionof L-selectin. Inhibition and blocking are also intended to include anymeasurable decrease in the binding affinity of IL-8 when in contact withan anti-IL-8 antibody as compared to IL-8 not in contact with ananti-IL-8 antibody, e.g., the blocking of binding of IL-8 to itsreceptor by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,99%, or 100%.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.Accordingly, the term “human monoclonal antibody” refers to antibodiesdisplaying a single binding specificity which have variable and constantregions derived from human germline immunoglobulin sequences. In oneembodiment, the human monoclonal antibodies are produced by a hybridomawhich includes a B cell obtained from a transgenic non-human animal,e.g., a transgenic mouse, having a genome comprising a human heavy chaintransgene and a light chain transgene fused to an immortalized cell.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created orisolated by recombinant means, such as (a) antibodies isolated from ananimal (e.g., a mouse) that is transgenic or transchromosomal for humanimmunoglobulin genes or a hybridoma prepared therefrom (describedfurther in Section I, below), (b) antibodies isolated from a host celltransformed to express the antibody, e.g., from a transfectoma, (c)antibodies isolated from a recombinant, combinatorial human antibodylibrary, and (d) antibodies prepared, expressed, created or isolated byany other means that involve splicing of human immunoglobulin genesequences to other DNA sequences. Such recombinant human antibodies havevariable and constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, a “heterologous antibody” is defined in relation to thetransgenic non-human organism producing such an antibody. This termrefers to an antibody having an amino acid sequence or an encodingnucleic acid sequence corresponding to that found in an organism notconsisting of the transgenic non-human animal, and generally from aspecies other than that of the transgenic non-human animal.

An “isolated antibody,” as used herein, is intended to refer to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities (e.g., an isolated antibody that bindsto IL-8 is substantially free of antibodies that bind antigens otherthan IL-8). An isolated antibody that binds to an epitope, isoform orvariant of human IL-8 may, however, have cross-reactivity to otherrelated antigens, e.g., from other species (e.g., IL-8 specieshomologs). Moreover, an isolated antibody may be substantially free ofother cellular material and/or chemicals. In one embodiment of theinvention, a combination of “isolated” monoclonal antibodies havingdifferent specificities are combined in a well defined composition.

As used herein, “specific binding” refers to antibody binding to apredetermined antigen. Typically, the antibody binds with an affinitycorresponding to a K_(D) of about 10⁻⁸ M or less, and binds to thepredetermined antigen with an affinity (as expressed by K_(D)) that isat least 10 fold less, and preferably at least 100 fold less than itsaffinity for binding to a non-specific antigen (e.g., BSA, casein) otherthan the predetermined antigen or a closely-related antigen.Alternatively, the antibody can bind with an affinity corresponding to aK_(A) of about 10⁷ M⁻¹ or higher, and binds to the predetermined antigenwith an affinity (as expressed by K_(A)) that is at least 10 foldhigher, and preferably at least 100 fold higher than its affinity forbinding to a non-specific antigen (e.g., BSA, casein) other than thepredetermined antigen or a closely-related antigen. The phrases “anantibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen”.

The term “k_(d)” (sec⁻¹), as used herein, is intended to refer to thedissociation rate constant of a particular antibody-antigen interaction.Said value is also referred to as the k_(off) value.

The term “k_(D)” (M⁻¹×sec⁻¹), as used herein, is intended to refer tothe association rate constant of a particular antibody-antigeninteraction.

The term “K_(A)” (M), as used herein, is intended to refer to theassociation equilibrium constant of a particular antibody-antigeninteraction.

The term “K_(D)” (M⁻¹), as used herein, is intended to refer to thedissociation equilibrium constant of a particular antibody-antigeninteraction.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes.

As used herein, “isotype switching” refers to the phenomenon by whichthe class, or isotype, of an antibody changes from one Ig class to oneof the other Ig classes.

As used herein, “nonswitched isotype” refers to the isotypic class ofheavy chain that is produced when no isotype switching has taken place;the C_(H) gene encoding the nonswitched isotype is typically the firstC_(H) gene immediately downstream from the functionally rearranged VDJgene. Isotype switching has been classified as classical ornon-classical isotype switching. Classical isotype switching occurs byrecombination events which involve at least one switch sequence regionin the transgene. Non-classical isotype switching may occur by, forexample, homologous recombination between human σ_(μ) and human Σ_(μ)(δ-associated deletion). Alternative non-classical switching mechanisms,such as intertransgene and/or interchromosomal recombination, amongothers, may occur and effectuate isotype switching.

As used herein, the term “switch sequence” refers to those DNA sequencesresponsible for switch recombination. A “switch donor” sequence,typically a μ switch region, will be 5′ (i.e., upstream) of theconstruct region to be deleted during the switch recombination. The“switch acceptor” region will be between the construct region to bedeleted and the replacement constant region (e.g., γ, ε, etc.).

As used herein, “glycosylation pattern” is defined as the pattern ofcarbohydrate units that are covalently attached to a protein, morespecifically to an immunoglobulin protein. A glycosylation pattern of aheterologous antibody can be characterized as being substantiallysimilar to glycosylation patterns which occur naturally on antibodiesproduced by the species of the non-human transgenic animal, when one ofordinary skill in the art would recognize the glycosylation pattern ofthe heterologous antibody as being more similar to said pattern ofglycosylation in the species of the non-human transgenic animal than tothe species from which the C_(H) genes of the transgene were derived.

The term “naturally-occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring.

The term “rearranged” as used herein refers to a configuration of aheavy chain or light chain immunoglobulin locus wherein a V segment ispositioned immediately adjacent to a D-J or J segment in a conformationencoding essentially a complete V_(H) or V_(L) domain, respectively. Arearranged immunoglobulin gene locus can be identified by comparison togermline DNA; a rearranged locus will have at least one recombinedheptamer/nonamer homology element.

The term “unrearranged” or “germline configuration” as used herein inreference to a V segment refers to the configuration wherein the Vsegment is not recombined so as to be immediately adjacent to a D or Jsegment.

The term “nucleic acid molecule”, as used herein, is intended to includeDNA molecules and RNA molecules. A nucleic acid molecule may besingle-stranded or double-stranded, but preferably is double-strandedDNA.

The term “isolated nucleic acid molecule,” as used herein in referenceto nucleic acids encoding intact antibodies or antibody portions (e.g.,V_(H), V_(L), CDR3) that bind to IL-8, is intended to refer to a nucleicacid molecule in which the nucleotide sequences encoding the intactantibody or antibody portion are free of other nucleotide sequencesencoding intact antibodies or antibody portions that bind antigens otherthan IL-8, which other sequences may naturally flank the nucleic acid inhuman genomic DNA. In one embodiment, the human anti-IL-8 antibodyincludes the amino acid sequence of 10F8, as well as heavy chain (V_(H))and light chain (V_(L)) variable amino acid regions having the sequencesshown in SEQ ID NOs:12 and 8 or encoded by the nucleotide sequencesshown in SEQ ID NOs: 10 and 6.

The present invention also encompasses “derivatives” of the amino acidsequences as set forth in SEQ ID NO: 8 or 12, wherein one or more of theamino acid residues have been derivatised, e.g., by acylation orglycosylation, without significantly affecting or altering the bindingcharacteristics of the antibody containing the amino acid sequences.

Furthermore, the present invention comprises antibodies in which one ormore alterations have been made in the Fc region in order to changefunctional or pharmacokinetic properties of the antibodies. Suchalterations may result in a decrease or increase of C1q binding and CDC(complement dependent cytotixicity) or of FcγR binding andantibody-dependent cellular cytotoxicity (ADCC). Substitutions can forexample be made in one or more of the amino acid positions 234, 235,236, 237, 297, 318, 320, and 322 of the heavy chain constant region,thereby causing an alteration in an effector function while retainingbinding to antigen as compared with the unmodified antibody, cf. U.S.Pat. No. 5,624,821 and U.S. Pat. No. 5,648,260. Further reference may behad to WO 00/42072 disclosing antibodies with altered Fc regions thatincrease ADCC, and WO 94/29351 disclosing antibodies having mutations inthe N-terminal region of the CH2 domain that alter the ability of theantibodies to bind to FcRI and thereby decreases the ability of theantibodies to bind to C1q which in turn decreases the ability of theantibodies to fix complement. Furthermore, Shields et al., J. Biol.Chem. (2001) 276:6591-6604 teaches combination variants, e.g.,T256A/S298A, S298A/E333A, and S298A/E333A/K334A, that improve FcγRIIIbinding.

The in vivo half-life of the antibodies can also be improved bymodifying the salvage receptor epitope of the Ig constant domain or anIg-like constant domain such that the molecule does not comprise anintact CH2 domain or an intact Ig Fc region, cf. U.S. Pat. No. 6,121,022and U.S. Pat. No. 6,194,551. The in vivo half-life can furthermore beincreased by making mutations in the Fc region, e.g., by substitutingthreonine for leucine at position 252, threonine for serine at position254, or threonine for phenylalanine at position 256, cf. U.S. Pat. No.6,277,375.

Furthermore, the glycosylation pattern of the antibodies can be modifiedin order to change the effector function of the antibodies. For example,the antibodies can be expressed in a transfectoma which does not add thefucose unit normally attached to the carbohydrate attached to Asn atposition 297 of Fc in order to enhance the affinity of Fc for FcγRIIIwhich in turn will result in an increased ADCC of the antibodies in thepresence of NK cells, cf. Shield et al. (2002) J. Biol. Chem.,277:26733. Furthermore, modification of galactosylation can be made inorder to modify CDC. Further reference may be had to WO 99/54342 andUmana et al., Nat. Biotechnol. (1999) 17:176 disclosing a CHO cell lineengineered to express GntIII resulting in the expression of monoclonalantibodies with altered glycoforms and improved ADCC activity.

Furthermore, the antibody fragments, e.g., Fab fragments, of theinvention can be pegylated to increase the half-life. This can becarried out by pegylation reactions known in the art, as described, forexample, in Focus on Growth Factors (1992) 3:4-10, EP 154 316 and EP 401384.

Accordingly, the invention includes antibodies encoded by the (heavy andlight chain variable region) nucleotide sequences disclosed hereinand/or containing the (heavy and light chain variable region) amino acidsequences disclosed herein (i.e., SEQ ID NOs: 10, 6, 12, and 8).

For nucleic acid and amino acid sequences, the term “homology” indicatesthe degree of identity between two sequences, when optimally aligned andcompared, with appropriate insertions or deletions. Alternatively,substantial homology exists when the DNA segments will hybridize underselective hybridization conditions, to the complement of the strand.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. The comparison of sequencesand determination of percent identity between two sequences can beaccomplished using a mathematical algorithm, as described in thenon-limiting examples below.

The percent identity between two nucleotide sequences can be determinedusing the GAP program in the GCG software package (available on thewebsite for Accelrys GCG, gcg.com), using a NWSgapdna.CMP matrix and agap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4,5, or 6. The percent identity between two nucleotide or amino acidsequences can also determined using the algorithm of E. Meyers and W.Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has beenincorporated into the ALIGN program (version 2.0), using a PAM120 weightresidue table, a gap length penalty of 12 and a gap penalty of 4. Inaddition, the percent identity between two amino acid sequences can bedetermined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453(1970)) algorithm which has been incorporated into the GAP program inthe GCG software package (available on the website for Accelrys GCG,gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gapweight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4,5, or 6.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify related sequences. Such searches canbe performed using the NBLAST and XBLAST programs (version 2.0) ofAltschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotidesearches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See the website of the National Center for BiotechnologyInformation (NCBI), National Library of Medicine, Building 38A,Bethesda, Md. 20894, U.S.A., ncbi.nlm.nih.gov.

The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components or other contaminants, e.g., other cellularnucleic acids or proteins, by standard techniques, includingalkaline/SDS treatment, CsCl banding, column chromatography, agarose gelelectrophoresis and others well known in the art. See, F. Ausubel, etal., ed. Current Protocols in Molecular Biology, Greene Publishing andWiley Interscience, New York (1987).

The nucleic acid compositions of the present invention, while often in anative sequence (except for modified restriction sites and the like),from either cDNA, genomic or mixtures thereof may be mutated inaccordance with standard techniques to provide gene sequences. Forcoding sequences, these mutations, may affect amino acid sequence asdesired. In particular, DNA sequences substantially homologous to orderived from native V, D, J, constant, switches and other such sequencesdescribed herein are contemplated (where “derived” indicates that asequence is identical or modified from another sequence).

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itaffects the transcription of the sequence. With respect to transcriptionregulatory sequences, operably linked means that the DNA sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in reading frame. For switch sequences, operablylinked indicates that the sequences are capable of effecting switchrecombination.

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby bereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which a recombinantexpression vector has been introduced. It should be understood that suchterms are intended to refer not only to the particular subject cell butalso to the progeny of such a cell. Because certain modifications mayoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. Recombinant host cells include, for example, CHO cells, andNS/0 cells.

The term “transfectoma”, as used herein, includes a recombinanteukaryotic host cell expressing the antibody, such as CHO cells, NS/0cells, HEK293 cells, plant cells, or fungi, including yeast cells.

As used herein, the term “subject” includes any human or non-humananimal. The term “non-human animal” includes all vertebrates, e.g.,mammals and non-mammals, such as non-human primates, sheep, dogs, cows,chickens, amphibians, reptiles, etc.

The terms “transgenic, non-human animal” refers to a non-human animalhaving a genome comprising one or more human heavy and/or light chaintransgenes or transchromosomes (either integrated or non-integrated intothe animal's natural genomic DNA) and which is capable of expressingfully human antibodies. For example, a transgenic mouse can have a humanlight chain transgene and either a human heavy chain transgene or humanheavy chain transchromosome, such that the mouse produces humananti-IL-8 antibodies when immunized with IL-8 antigen and/or cellsexpressing IL-8. The human heavy chain transgene can be integrated intothe chromosomal DNA of the mouse, as is the case for transgenic, e.g.,HuMAb mice, or the human heavy chain transgene can be maintainedextrachromosomally, as is the case for transchromosomal (e.g., KM) miceas described in WO 02/43478. Such transgenic and transchromosomal miceare capable of producing multiple isotypes of human monoclonalantibodies to IL-8 (e.g., IgG, IgA and/or IgE) by undergoing V-D-Jrecombination and isotype switching. Transgenic, non-human animals canalso be used for production of a specific anti-IL-8 antibody byintroducing genes encoding such specific anti-IL-8 antibody, for exampleby operatively linking the genes to a gene which is expressed in themilk of the animal.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. Production of Human Antibodies to IL-8

The human monoclonal antibodies of the invention can be produced by avariety of techniques, including conventional monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein (1975) Nature 256:495. Although somatic cellhybridization procedures are preferred, in principle, other techniquesfor producing monoclonal antibody can be employed e.g., viral oroncogenic transformation of B lymphocytes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

In a preferred embodiment, human monoclonal antibodies directed againstIL-8 can be generated using transgenic mice carrying parts of the humanimmune system rather than the mouse system. These transgenic mice,referred to herein as “HuMAb” mice, contain a human immunoglobulin geneminiloci that encodes unrearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous μ and κ chain loci (Lonberg, et al. (1994)Nature 368(6474):856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ light chain, and in response toimmunization, the introduced human heavy and light chain transgenesundergo class switching and somatic mutation to generate high affinityhuman IgGx monoclonal antibodies (Lonberg, N. et al. (1994), supra;reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. Vol.13:65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci.764:536-546). The preparation of HuMAb mice is described in detailSection II below and in Taylor, L. et al. (1992) Nucleic Acids Research20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656;Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi etal. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J.12:821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Lonberg,N. et al., (1994) Nature 368(6474):856-859; Lonberg, N. (1994) Handbookof Experimental Pharmacology 113:49-101; Taylor, L. et al. (1994)International Immunology 6:579-591; Lonberg, N. and Huszar, D. (1995)Intern. Rev. Immunol. Vol. 13:65-93; Harding, F. and Lonberg, N. (1995)Ann. N.Y. Acad. Sci. 764:536-546; Fishwild, D. et al. (1996) NatureBiotechnology 14:845-851. See further, U.S. Pat. Nos. 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016;5,814,318; 5,874,299; and 5,770,429; all to Lonberg, N. and Kay, R. M.and GenPharm International; U.S. Pat. No. 5,545,807 to Surani et al.;International Publication Nos. WO 98/24884, published on Jun. 11, 1998;WO 94/25585, published Nov. 10, 1994; WO 93/1227, published Jun. 24,1993; WO 92/22645, published Dec. 23, 1992; and WO 92/03918, publishedMar. 19, 1992. Preferred HuMAb mice have a JKD disruption in theirendogenous light chain (kappa) genes (as described in Chen et al. (1993)EMBO J. 12: 821-830), a CMD disruption in their endogenous heavy chaingenes (as described in Example 1 of WO 01/14424 by Korman et al.), aKCoS human kappa light chain transgene (as described in Fishwild et al.(1996) Nature Biotechnology 14:845-851), and a HCo7 human heavy chaintransgene (as described in U.S. Pat. No. 5,770,429 by Lonberg, N. andKay, R. M.) and/or a HCo12 human heavy chain transgene (as described inExample 2 of WO 01/14424 by Korman et al.).

Alternatively, mice carrying human immunoglobulin genes on atranschromosomic fragment can be used to generate anti-IL-8 antibodies.Preparation of such transchromosomic mice are described in WO 97/07671by Tomizuka et al. A preferred mouse is one in which certain humanimmunoglobulin genes are carried on a transgene and others are carriedon a transchromosome, such as a mouse carrying a human light chaintransgene (e.g., the KCoS kappa chain transgene) and a human heavy chaintranschromosome (e.g, the SC20 transchromosome) as described in detailin WO 02/43478 by Ishida et al.

HuMAb Immunizations

To generate fully human monoclonal antibodies to IL-8, HuMAb mice can beimmunized with a purified or enriched preparation of IL-8 antigen and/orcells producing IL-8 and/or recombinant IL-8, as described by Lonberg,N. et al. (1994) Nature 368(6474):856-859; Fishwild, D. et al. (1996)Nature Biotechnology 14:845-851 and WO 98/24884. Preferably, the micewill be 6-16 weeks of age upon the first infusion. For example,recombinant IL-8 can be used to immunize the HuMAb miceintraperitoneally.

Cumulative experience with various antigens has shown that the HuMAbtransgenic mice respond best when initially immunized intraperitoneally(IP) with antigen in complete Freund's adjuvant, followed by every otherweek i.p. immunizations (up to a total of 6) with antigen in incompleteFreund's adjuvant. The immune response can be monitored over the courseof the immunization protocol with plasma samples being obtained byretroorbital bleeds. The plasma can be screened by ELISA (as describedbelow), and mice with sufficient titers of anti-IL-8 humanimmunoglobulin can be used for fusions. Mice can be boostedintravenously with antigen 3 days before sacrifice and removal of thespleen. It is expected that 2-3 fusions for each antigen may need to beperformed. Several mice will be immunized for each antigen. For example,a total of twelve HuMAb mice of the HCo7 and HCo12 strains can beimmunized.

Generation of Hybridomas Producing Human Monoclonal Antibodies to IL-8

The mouse splenocytes can be isolated and fused with PEG to a mousemyeloma cell line based upon standard protocols. The resultinghybridomas are then screened for the production of antigen-specificantibodies. For example, single cell suspensions of splenic lymphocytesfrom immunized mice are fused to one-sixth the number of P3X63-Ag8.653nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG. Cellsare plated at approximately 2×10⁵ in flat bottom microtiter plates,followed by a two week incubation in selective medium containing 20%fetal Clone Serum, 18% “653” conditioned media, 5% origen (IGEN), 4 mML-glutamine, 1 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055mM 2-mercaptoethanol, 50 units/mL penicillin, 50 mg/mL streptomycin, 50mg/mL gentamycin and 1×HAT (Sigma; the HAT is added 24 hours after thefusion). After two weeks, cells are cultured in medium in which the HATis replaced with HT. Individual wells are then screened by ELISA forhuman anti-IL-8 monoclonal IgM and IgG antibodies. Once extensivehybridoma growth occurs, medium is observed usually after 10-14 days.The antibody secreting hybridomas are replated, screened again, and ifstill positive for human IgG, anti-IL-8 monoclonal antibodies, can besubcloned at least twice by limiting dilution. The stable subclones arethen cultured in vitro to generate small amounts of antibody in tissueculture medium for characterization.

Generation of Transfectomas Producing Human Monoclonal Antibodies toIL-8

Human antibodies of the invention also can be produced in a host celltransfectoma using, for example, a combination of recombinant DNAtechniques and gene transfection methods as is well known in the art(Morrison, S. (1985) Science 229:1202).

For example, to express the antibodies, or antibody fragments thereof,DNAs encoding partial or full-length light and heavy chains, can beobtained by standard molecular biology techniques (e.g., PCRamplification, site directed mutagenesis) and can be inserted intoexpression vectors such that the genes are operatively linked totranscriptional and translational control sequences. In this context,the term “operatively linked” is intended to mean that an antibody geneis ligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by standard methods (e.g., ligation of complementary restrictionsites on the antibody gene fragment and vector, or blunt end ligation ifno restriction sites are present). The light and heavy chain variableregions of the antibodies described herein can be used to createfull-length antibody genes of any antibody isotype by inserting theminto expression vectors already encoding heavy chain constant and lightchain constant regions of the desired isotype such that the V_(H)segment is operatively linked to the C_(H) segment(s) within the vectorand the V_(L) segment is operatively linked to the C_(L) segment withinthe vector. Additionally or alternatively, the recombinant expressionvector can encode a signal peptide that facilitates secretion of theantibody chain from a host cell. The antibody chain gene can be clonedinto the vector such that the signal peptide is linked in-frame to theamino terminus of the antibody chain gene. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide (i.e., asignal peptide from a non-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the invention carry regulatory sequences that control theexpression of the antibody chain genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the antibody chain genes.Such regulatory sequences are described, for example, in Goeddel; GeneExpression Technology. Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). It will be appreciated by those skilled in the artthat the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. Alternatively,nonviral regulatory sequences may be used, such as the ubiquitinpromoter or β-globin promoter.

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the invention may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Preferred selectable marker genes includethe dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell bystandard techniques. The various forms of the term “transfection” areintended to encompass a wide variety of techniques commonly used for theintroduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is theoreticallypossible to express the antibodies of the invention in eitherprokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells, and most preferably mammalian host cells, is the mostpreferred because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Preferred mammalian host cells for expressing the recombinant antibodiesof the invention include CHO cells (including dhfr-CHO cells, describedin Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220,used with a DHFR selectable marker, e.g., as described in R. J. Kaufmanand P. A. Sharp (1982) Mol. Biol. 159:601-621), NS/0 myeloma cells, COScells, HEK293 cells and SP2.0 cells. In particular for use with NS/0myeloma cells, another preferred expression system is the GS (glutaminesynthetase) gene expression system disclosed in WO 87/04462, WO 89/01036and EP 338 841. When recombinant expression vectors encoding antibodygenes are introduced into mammalian host cells, the antibodies areproduced by culturing the host cells for a period of time sufficient toallow for expression of the antibody in the host cells or, morepreferably, secretion of the antibody into the culture medium in whichthe host cells are grown. Antibodies can be recovered from the culturemedium using standard protein purification methods.

Further Recombinant Means for Producing Human Monoclonal Antibodies toIL-8

Alternatively the cloned antibody genes can be expressed in otherexpression systems, including prokaryotic cells, such as microorganisms,e.g., E. coli for the production of scFv antibodies, algi, as well asinsect cells. Furthermore, the antibodies can be produced in transgenicnon-human animals, such as in milk from sheep and rabbits or eggs fromhens, or in transgenic plants. See, e.g., Verma, R., et al. (1998).Antibody engineering: Comparison of bacterial, yeast, insect andmammalian expression systems. J. Immunol. Meth. 216:165-181; Pollock, etal. (1999). Transgenic milk as a method for the production ofrecombinant antibodies. J. Immunol. Meth. 231:147-157; and Fischer, R.,et al. (1999). Molecular farming of recombinant antibodies in plants.Biol. Chem. 380:825-839.

Use of Partial Antibody Sequences to Express Intact Antibodies

Antibodies interact with target antigens predominantly through aminoacid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998, Nature332:323-327; Jones, P. et al., 1986, Nature 321:522-525; and Queen, C.et al., 1989, Proc. Natl. Acad. See. U.S.A. 86:10029-10033). Suchframework sequences can be obtained from public DNA databases thatinclude germline antibody gene sequences. These germline sequences willdiffer from mature antibody gene sequences because they will not includecompletely assembled variable genes, which are formed by V(D)J joiningduring B cell maturation. Germline gene sequences will also differ fromthe sequences of a high affinity secondary repertoire antibody whichcontains mutations throughout the variable gene but typically clusteredin the CDRs. For example, somatic mutations are relatively infrequent inthe amino terminal portion of framework region 1 and in thecarboxy-terminal portion of framework region 4. Furthermore, manysomatic mutations do not significantly alter the binding properties ofthe antibody. For this reason, it is not necessary to obtain the entireDNA sequence of a particular antibody in order to recreate an intactrecombinant antibody having binding properties similar to those of theoriginal antibody (see WO 99/45962). Partial heavy and light chainsequence spanning the CDR regions is typically sufficient for thispurpose. The partial sequence is used to determine which germlinevariable and joining gene segments contributed to the recombinedantibody variable genes. The germline sequence is then used to fill inmissing portions of the variable regions. Heavy and light chain leadersequences are cleaved during protein maturation and do not contribute tothe properties of the final antibody. To add missing sequences, clonedcDNA sequences can be combined with synthetic oligonucleotides byligation or PCR amplification. Alternatively, the entire variable regioncan be synthesized as a set of short, overlapping, oligonucleotides andcombined by PCR amplification to create an entirely synthetic variableregion clone. This process has certain advantages such as elimination orinclusion of particular restriction sites, or optimization of particularcodons.

The nucleotide sequences of heavy and light chain transcripts from ahybridoma are used to design an overlapping set of syntheticoligonucleotides to create synthetic V sequences with identical aminoacid coding capacities as the natural sequences. The synthetic heavy andkappa chain sequences can differ from the natural sequences in threeways: strings of repeated nucleotide bases are interrupted to facilitateoligonucleotide synthesis and PCR amplification; optimal translationinitiation sites are incorporated according to Kozak's rules (Kozak(1991) J. Biol. Chem. 266:19867-19870); and, HindIII sites areengineered upstream of the translation initiation sites.

For both the heavy and light chain variable regions, the optimizedcoding, and corresponding non-coding, strand sequences are broken downinto 30-50 nucleotide approximately the midpoint of the correspondingnon-coding oligonucleotide. Thus, for each chain, the oligonucleotidescan be assembled into overlapping double stranded sets that spansegments of 150-400 nucleotides. The pools are then used as templates toproduce PCR amplification products of 150-400 nucleotides. Typically, asingle variable region oligonucleotide set will be broken down into twopools which are separately amplified to generate two overlapping PCRproducts. These overlapping products are then combined by PCRamplification to form the complete variable region. It may also bedesirable to include an overlapping fragment of the heavy or light chainconstant region (including the BbsI site of the kappa light chain, orthe AgeI site of the gamma heavy chain) in the PCR amplification togenerate fragments that can easily be cloned into the expression vectorconstructs.

The reconstructed heavy and light chain variable regions are thencombined with cloned promoter, leader sequence, translation initiation,constant region, 3′ untrans-lated, polyadenylation, and transcriptiontermination sequences to form expression vector constructs. The heavyand light chain expression constructs can be combined into a singlevector, co-transfected, serially transfected, or separately transfectedinto host cells which are then fused to form a host cell expressing bothchains.

Plasmids for use in construction of expression vectors for human IgGκare described below. The plasmids were constructed so that PCR amplifiedV heavy and V kappa light chain cDNA sequences could be used toreconstruct complete heavy and light chain minigenes. These plasmids canbe used to express completely human IgG1,κ or IgG4,κ antibodies. Similarplasmids can be constructed for expression of other heavy chainisotypes, or for expression of antibodies comprising lambda lightchains.

Thus, in another aspect of the invention, the structural features of ahuman anti-IL-8 antibody of the invention, e.g., 10F8, are used tocreate structurally related human anti-IL-8 antibodies that retain atleast one functional property of the antibodies of the invention, suchas binding to IL-8. More specifically, one or more CDRs of 10F8 can becombined recombinantly with known human framework regions and CDRs tocreate additional, recombinantly-engineered, human anti-IL-8 antibodiesof the invention.

Accordingly, in another embodiment, the invention provides a method forpreparing an anti-IL-8 antibody comprising:

preparing an antibody comprising (1) human heavy chain framework regionsand human heavy chain CDRs, wherein at least one of the human heavychain CDRs comprises an amino acid sequence selected from the amino acidsequences of CDRs shown in FIG. 5 (SEQ ID NOs: 22, 23, or 24); and (2)human light chain framework regions and human light chain CDRs, whereinat least one of the light chain CDRs comprises an amino acid sequenceselected from the amino acid sequences of CDRs shown in FIG. 3 (SEQ IDNOs: 16, 17, or 18);

wherein the antibody retains the ability to bind to IL-8.

The ability of the antibody to bind IL-8 can be determined usingstandard binding assays, such as those set forth in the Examples (e.g.,an ELISA).

Since it is well known in the art that antibody heavy and light chainCDR3 domains play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen, the recombinantantibodies of the invention prepared, as set forth above, preferablycomprise the heavy and light chain CDR3s of 10F8. The antibodies furthercan comprise the CDR2s of 10F8. The antibodies further can comprise theCDR1s of 10F8. The antibodies can further comprise any combinations ofthe CDRs.

Accordingly, in another embodiment, the invention further providesanti-IL-8 antibodies comprising: (1) human heavy chain frameworkregions, a human heavy chain CDR1 region, a human heavy chain CDR2region, and a human heavy chain CDR3 region, wherein the human heavychain CDR3 region is the heavy chain CDR3 of 10F8 as shown in FIG. 5(SEQ ID NO: 24); and (2) human light chain framework regions, a humanlight chain CDR1 region, a human light chain CDR2 region, and a humanlight chain CDR3 region, wherein the human light chain CDR3 region isthe light chain CDR3 of 10F8 as shown in FIG. 3 (SEQ ID NO: 18), whereinthe antibody binds IL-8. The antibody may further comprise the heavychain CDR2 and/or the light chain CDR2 of 10F8. The antibody may furthercomprise the heavy chain CDR1 and/or the light chain CDR1 of 10F8.

The CDR1, 2, and/or 3 regions of the engineered antibodies describedabove can comprise the exact amino acid sequence(s) as those of 10F8disclosed herein. However, the ordinarily skilled artisan willappreciate that some deviation from the exact CDR sequences of 10F8 maybe possible while still retaining the ability of the antibody to bindIL-8 effectively. Accordingly, in another embodiment, the engineeredantibody may be composed of one or more CDRs that are 95%, 98% or 99.5%identical to one or more CDRs of 10F8.

Accordingly, in another embodiment, the invention provides anti-IL-8antibodies comprising a heavy chain variable region and/or a light chainvariable region which is homologous to or derived from its correspondinggermline variable region sequence, e.g., the Vκ A-27 germline nucleotideand amino acid sequences shown in FIGS. 2 and 3 (SEQ ID NOs:5 and 7,respectively) and/or the V_(H) 3-33 germline nucleotide and amino acidsequences shown in FIGS. 4 and 5 (SEQ ID NOs:9 and 11, respectively),and retains at least one functional property of the antibodies of theinvention, such as binding to IL-8.

Other particular antibodies of the invention bind to human IL-8 andcomprise a light chain variable region having an amino acid sequencewhich is at least about 94% identical, preferably about 96%, morepreferably about 97%, 98%, or 99% identical to the germline amino acidsequence shown in FIG. 3 (SEQ ID NO:7) and/or a heavy chain variableregion having an amino acid sequence which is at least about 92%identical, preferably about 94%, more preferably about 96%, 97%, 98%, or99% identical to the amino acid sequence shown in FIG. 5 (SEQ ID NO:11).Alternatively, the antibodies may comprise a light chain variable regionencoded by a nucleotide sequence which is at least about 94% identical,preferably about 96%, more preferably about 97%, 98%, or 99% identicalto the germline nucleotide sequence shown in FIG. 2 (SEQ ID NO:5) and/ora heavy chain variable region encoded by a nucleotide sequence which isat least about 92% identical, preferably about 94%, more preferablyabout 96%, 97%, 98%, or 99% identical to the nucleotide sequence shownin FIG. 4 (SEQ ID NO:9).

Particular antibodies of the invention also include human antibodieswhich bind to human IL-8 and comprise a light chain variable regionderived from the Vκ A-27 germline amino acid sequence as shown in FIG. 3(SEQ ID NO:7) and have an amino acid sequence which comprises at leastone residue selected from the group consisting of an isoleucine atposition 29, a proline residue at position 52, an alanine residue atposition 93, a glycine residue at position 94, a leucine residue atposition 96, a proline residue at position 100, an aspartic acid atposition 105, as shown in FIG. 3, and any combination thereof.Alternatively or in addition, the antibodies may comprise a heavy chainvariable region derived from the V_(H) 3-33 germline amino acid sequenceas shown in FIG. 5 (SEQ ID NO:11) and have an amino acid sequence whichcomprises at least one residue selected from the group consisting of anglutamine at position 3, a histidine residue at position 31, a tyrosineresidue at position 35, an isoleucine residue at position 51, a tyrosineresidue at position 57, an asparagine residue at position 60, an alanineresidue at position 61, an isoleucine residue at position 70, anasparagine residue at position 74, a glutamine residue at position 82,an arginine residue at position 100, a leucine residue at position 103,as shown in FIG. 5, and any combination thereof.

In another embodiment, the invention provides human anti-IL-8 antibodieswhich comprise a CDR domain having a human heavy and light chain CDR1region, a human heavy and light chain CDR2 region, and a human heavy andlight chain CDR3 region, wherein the CDR1, CDR2, and CDR3 heavy chainregions are derived from the V_(H) 3-33 germline amino acid sequence asshown in FIG. 5 (SEQ ID NO:11) or wherein the CDR1, CDR2, and CDR3 lightchain regions are derived from the Vκ A-27 germline amino acid sequenceas shown in FIG. 3 (SEQ ID NO:7), and wherein

(a) the CDR1 human heavy chain region comprises a histidine and atyrosine residue at positions 1 and 5, respectively, as shown in FIG. 5(SEQ ID NO:22);

(b) the CDR2 human heavy chain region comprises an isoleucine, tyrosine,asparagine, and alanine residue at positions 2, 8, 11, and 12,respectively, as shown in FIG. 5 (SEQ ID NO:23);

(c) the CDR3 human heavy chain region comprises an arginine and leucineresidue at positions 2 and 5, respectively, as shown in FIG. 5 (SEQ IDNO:24);

(d) the CDR1 human light chain region comprises an isoleucine residue atposition 6, as shown in FIG. 3 (SEQ ID NO:16);

(e) the CDR2 human light chain region comprises a proline residue atposition 2, as shown in FIG. 3 (SEQ ID NO:17);

(f) the CDR3 human light chain region comprises a tyrosine, alanine,glycine, and leucine residue at positions 3, 4, 5, and 6, respectively,as shown in FIG. 3 (SEQ ID NO:18); and

(g) any combination of (a), (b), (c), (d), (e), or (f).

In another embodiment, the human antibodies may comprise a human heavyand light chain CDR1 region, a human heavy and light chain CDR2 region,and a human heavy and light chain CDR3 region, wherein the CDR1, CDR2,and CDR3 heavy chain regions are derived from the V_(H) 3-33 germlineamino acid sequence as shown in FIG. 5 (SEQ ID NO:11) and/or wherein theCDR1, CDR2, and CDR3 light chain regions are derived from the Vκ A-27germline amino acid sequence as shown in FIG. 3 (SEQ ID NO:7), andwherein at least one of the CDR domains is selected from the groupconsisting of:

(a) a light chain CDR1 region comprising an amino acid sequence which isat least 92% identical to the amino acid sequence shown in FIG. 3 (SEQID NO:16);

(b) a light chain CDR2 region comprising an amino acid sequence which isat least 86% identical to the amino acid sequence shown in FIG. 3 (SEQID NO:17);

(c) a light chain CDR3 region comprising an amino acid sequence which isat least 43% identical to the amino acid sequence shown in FIG. 3 (SEQID NO:18);

(d) a heavy chain CDR1 region comprising an amino acid sequence which isat least 61% identical to the amino acid sequence shown in FIG. 5 (SEQID NO:22);

(e) a heavy chain CDR2 region comprising an amino acid sequence which isat least 77% identical to the amino acid sequence shown in FIG. 5 (SEQID NO:23); and

(f) a heavy chain CDR3 region comprising an amino acid sequence which isat least 76% identical to the amino acid sequence shown in FIG. 5 (SEQID NO:24); and (g) any combination of (a), (b), (c), (d), (e), or (f).

In yet another embodiment, at least one of the CDR domains of the humanantibodies comprises an amino acid sequence selected from the groupconsisting of:

(a) the amino acid sequence of the light chain CDR1 region comprises anisoleucine residue at position 6, as shown in FIG. 3 (SEQ ID NO:16);

(b) the amino acid sequence of the light chain CDR2 region comprises aproline residue at position 2, as shown in FIG. 3 (SEQ ID NO:17);

(c) the amino acid sequence of the light chain CDR3 region comprises atyrosine residue at position 3, an alanine residue at position 4, aglycine residue at position 5, and a leucine residue at position 7, asshown in FIG. 3 (SEQ ID NO:18);

(d) the amino acid sequence of the heavy chain CDR1 region comprises ahistidine residue at position 1, and a tyrosine residue at position 5,as shown in FIG. 5 (SEQ ID NO:22);

(e) the amino acid sequence of the heavy chain CDR2 region comprises anisoleucine residue at position 2, a tyrosine residue at position 8, anasparagine residue at position 11, and an alanine residue at position12, as shown in FIG. 5 (SEQ ID NO:23);

(f) the amino acid sequence of the heavy chain CDR3 region comprises anarginine residue at position 2 and a leucine residue at position 5, asshown FIG. 5 (SEQ ID NO:24); and

(g) any combination of (a), (b), (c), (d), (e), or (f).

In another embodiment the invention relates to an isolated humanmonoclonal antibody which binds to human IL-8 comprising a V_(L) CDR3domain having the amino acid sequence:

Gln-Gln-Tyr-X₁-X₂-Ser-X₃-Thr (SEQ ID NO: 46),

wherein X₁, X₂ and X₃ each represents a natural amino acid residue, andX₁ is different from Gly, or X₂ is different from Ser, or X₃ isdifferent from Pro.

In one embodiment X₁ is different from Gly, X₂ is different from Ser,and X₃ is different from Pro.

In a further embodiment X₁ is Ala, and X₂ and X₃ are independently Gly,Ala, Val, Leu, or Ile.

In yet another embodiment the invention relates to an isolated humanmonoclonal antibody which binds to human IL-8 comprising a V_(H) CDR3domain having the amino acid sequence:

Asp-X₄-Val-Gly-X₅-Phe-Asp-Tyr (SEQ ID NO: 47),

wherein X₄ is Lys, Arg, or His, and X₅ is Gly, Ala, Val, Leu, or Ile.

In still another embodiment the invention relates to an isolated humanmonoclonal antibody which binds to human IL-8 comprising a V_(L) CDR3domain as disclosed in the above embodiments and a V_(H) CDR3 domain asdisclosed in the above embodiments.

In addition, or alternative, to simply binding IL-8, antibodies such asthose described above may be selected for their retention of otherfunctional properties of antibodies of the invention, such as:

(1) binding to human IL-8 and inhibiting IL-8 induced proinflammatoryeffects;

(2) inhibiting binding of IL-8 to its receptors on neutrophils;

(3) inhibiting IL-8 induced chemotactic activity for neutrophils;

(4) inhibiting IL-8 induced calcium flux;

(5) inhibiting IL-8 induced changes in expression levels of adhesionmolecules on neutrophils;

(6) binding to human IL-8 and inhibiting IL-8 induced increasedexpression of CD11b (Mac-1) and decreased expression of L-selectin onneutrophils;

(7) not cross-reacting with related chemokines, such as human GRO-α,human GRO-β, human IP-10 and human NAP-2;

(8) binding to human IL-8 with a dissociation equilibrium constant(K_(D)) of approximately 10⁻⁸ M or less, such as 10⁻⁹ M or less, 10⁻¹⁹ Mor less, or 10⁻¹¹ M or even less; and/or

(9) significantly inhibiting chemotaxis induced by biological fluidswhich contain multiple chemotactic factors including IL8.

Characterization of Binding of Human Monoclonal Antibodies to IL-8

To characterize binding of human monoclonal IL-8 antibodies of theinvention, sera from immunized mice can be tested, for example, byELISA. In a typical (but non-limiting) example of an ELISA protocol,microtiter plates are coated with purified IL-8 at 0.25 μg/mL in PBS,and then blocked with 5% bovine serum albumin (BSA) in PBS. Dilutions ofplasma from IL-8-immunized mice are added to each well and incubated for1-2 hours at 37° C. The plates are washed with PBS/Tween and thenincubated with a goat-anti-human IgG Fc-specific polyclonal reagentconjugated to alkaline phosphatase for 1 hour at 37° C. After washing,the plates are developed with pNPP substrate (1 mg/mL), and analyzed atOD of 405-650. Preferably, mice which develop the highest titers will beused for fusions.

An ELISA assay as described above can also be used to screen forhybridomas that show positive reactivity with IL-8 immunogen. Hybridomasthat bind with high avidity to IL-8 will be subcloned and furthercharacterized. One clone from each hybridoma, which retains thereactivity of the parent cells (by ELISA), can be chosen for making a5-10 vial cell bank stored at −140° C., and for antibody purification.

To purify human anti-IL-8 antibodies, selected hybridomas can be grownin two-liter spinner-flasks for monoclonal antibody purification.Supernatants can be filtered and concentrated before affinitychromatography with protein A-sepharose (Pharmacia, Piscataway, N.J.).Eluted IgG can be checked by gel electrophoresis and high performanceliquid chromatography to ensure purity. The buffer solution can beexchanged into PBS, and the concentration can be determined by OD₂₈₀using 1.43 extinction coefficient. The monoclonal antibodies can bealiquoted and stored at −80° C.

To determine if the selected human anti-IL-8 monoclonal antibodies bindto unique epitopes, site-directed or multi-site directed mutagenesis canbe used.

To determine the isotype of purified antibodies, isotype ELISAs can beperformed. For example, wells of microtiter plates can be coated with 10μg/mL of anti-human Ig overnight at 4° C. After blocking with 5% BSA,the plates are reacted with 10 μg/mL of monoclonal antibodies orpurified isotype controls, at ambient temperature for two hours. Thewells can then be reacted with either human IgG1 or human IgM-specificalkaline phosphatase-conjugated probes. Plates are developed andanalyzed as described above.

Anti-IL-8 human IgGs can be tested for reactivity with IL-8 antigen byWestern blotting. For example, cell extracts from cells producing IL-8can be prepared and subjected to sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. After electrophoresis, the separatedantigens will be transferred to nitrocellulose membranes, blocked with20% mouse serum, and probed with the monoclonal antibodies to be tested.Human IgG binding can be detected using anti-human IgG alkalinephosphatase and developed with BCIP/NBT substrate tablets (Sigma Chem.Co., St. Louis, Mo.).

II. Production of Transgenic Non-Human Animals which Generate HumanMonoclonal Anti-IL-8 Antibodies

In yet another aspect, the invention provides transgenic andtranschromosomal non-human animals, such as transgenic ortranschromosomal mice, which are capable of expressing human antibodiesthat specifically bind to IL-8. In a particular embodiment, theinvention provides a transgenic or transchromosomal mouse having agenome comprising a human heavy chain transgene, such that the mouseproduces human anti-IL-8 antibodies when immunized with cells producingIL-8. The human heavy chain transgene can be integrated into thechromosomal DNA of the mouse, as is the case for transgenic, e.g., HuMAbmice, as described in detail herein and exemplified. Alternatively, thehuman heavy chain transgene can be maintained extrachromosomally, as isthe case for transchromosomal (e.g., KM) mice. More particularly, in theKM mouse strain, the endogenous mouse kappa light chain gene has beenhomozygously disrupted as described in Chen et al. (1993) EMBO J.12:811-820 and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of WO 01/09187. Thismouse strain carries a human kappa light chain transgene, KCoS, asdescribed in Fishwild et al. (1996) Nature Biotechnology 14:845-851.This mouse strain also carries a human heavy chain transchromosomecomposed of chromosome 14 fragment hCF (SC20) as described in WO02/43478.

Such transgenic and transchromosomal animals are capable of producingmultiple isotypes of human monoclonal antibodies to IL-8 (e.g., IgG, IgAand/or IgE) by undergoing V-D-J/V-J recombination and isotype switching.

The design of a transgenic or transchromosomal non-human animal thatresponds to foreign antigen stimulation with a heterologous antibodyrepertoire, requires that the heterologous immunoglobulin transgenescontained within the transgenic animal function correctly throughout thepathway of B cell development. This includes, for example, isotypeswitching of the heterologous heavy chain transgene. Accordingly,transgenes are constructed so as that isotype switching can be inducedand one or more of the following characteristics of antibody genes: (1)high level and cell-type specific expression, (2) functional generearrangement, (3) activation of and response to allelic exclusion, (4)expression of a sufficient primary repertoire, (5) signal transduction,(6) somatic hypermutation, and (7) domination of the transgene antibodylocus during the immune response.

Not all of the foregoing criteria need be met. For example, in thoseembodiments wherein the endogenous immunoglobulin loci of the transgenicanimal are functionally disrupted, the transgene need not activateallelic exclusion. Further, in those embodiments wherein the transgenecomprises a functionally rearranged heavy and/or light chainimmunoglobulin gene, the second criteria of functional generearrangement is unnecessary, at least for that transgene which isalready rearranged. For background on molecular immunology, see,Fundamental Immunology, 2nd edition (1989), Paul William E., ed. RavenPress, N.Y.

In certain embodiments, the transgenic or transchromosomal non-humananimals used to generate the human monoclonal antibodies of theinvention contain rearranged, unrearranged or a combination ofrearranged and unrearranged heterologous immunoglobulin heavy and lightchain transgenes in the germline of the transgenic animal. Each of theheavy chain transgenes comprises at least one C_(H) gene. In addition,the heavy chain transgene may contain functional isotype switchsequences, which are capable of supporting isotype switching of aheterologous transgene encoding multiple C_(H) genes in the B cells ofthe transgenic animal. Such switch sequences may be those which occurnaturally in the germline immunoglobulin locus from the species thatserves as the source of the transgene C_(H) genes, or such switchsequences may be derived from those which occur in the species that isto receive the transgene construct (the transgenic animal). For example,a human transgene construct that is used to produce a transgenic mousemay produce a higher frequency of isotype switching events if itincorporates switch sequences similar to those that occur naturally inthe mouse heavy chain locus, as presumably the mouse switch sequencesare optimized to function with the mouse switch recombinase enzymesystem, whereas the human switch sequences are not. Switch sequences maybe isolated and cloned by conventional cloning methods, or may besynthesized de novo from overlapping synthetic oligonucleotides designedon the basis of published sequence information relating toimmunoglobulin switch region sequences (Mills et al., Nucl. Acids Res.15:7305-7316 (1991); Sideras et al., Intl. Immunol. 1:631-642 (1989)).For each of the foregoing transgenic animals, functionally rearrangedheterologous heavy and light chain immunoglobulin transgenes are foundin a significant fraction of the B cells of the transgenic animal (atleast 10%).

The transgenes used to generate the transgenic non-human animals of theinvention include a heavy chain transgene comprising DNA encoding atleast one variable gene segment, one diversity gene segment, one joininggene segment and at least one constant region gene segment. Theimmunoglobulin light chain transgene comprises DNA encoding at least onevariable gene segment, one joining gene segment and at least oneconstant region gene segment. The gene segments encoding the light andheavy chain gene segments are heterologous to the transgenic animal inthat they are derived from, or correspond to, DNA encodingimmunoglobulin heavy and light chain gene segments from a species notconsisting of the transgenic non-human animal. In one aspect of theinvention, the transgene is constructed such that the individual genesegments are unrearranged, i.e., not rearranged so as to encode afunctional immunoglobulin light or heavy chain. Such unrearrangedtransgenes support recombination of the V, D, and J gene segments(functional rearrangement) and preferably support incorporation of allor a portion of a D region gene segment in the resultant rearrangedimmunoglobulin heavy chain within the transgenic animal when exposed toIL-8 antigen.

In an alternative embodiment, the transgenes comprise an unrearranged“minilocus”. Such transgenes typically comprise a substantial portion ofthe C, D, and J segments as well as a subset of the V gene segments. Insuch transgene constructs, the various regulatory sequences, e.g.,promoters, enhancers, class switch regions, splice-donor andsplice-acceptor sequences for RNA processing, recombination signals andthe like, comprise corresponding sequences derived from the heterologousDNA. Such regulatory sequences may be incorporated into the transgenefrom the same or a related species of the non-human animal used in theinvention. For example, human immunoglobulin gene segments may becombined in a transgene with a rodent immunoglobulin enhancer sequencefor use in a transgenic mouse. Alternatively, synthetic regulatorysequences may be incorporated into the transgene, wherein such syntheticregulatory sequences are not homologous to a functional DNA sequencethat is known to occur naturally in the genomes of mammals. Syntheticregulatory sequences are designed according to consensus rules, such as,for example, those specifying the permissible sequences of asplice-acceptor site or a promoter/enhancer motif. For example, aminilocus comprises a portion of the genomic immunoglobulin locus havingat least one internal (i.e., not at a terminus of the portion) deletionof a non-essential DNA portion (e.g., intervening sequence; intron orportion thereof) as compared to the naturally-occurring germline Iglocus.

Preferred transgenic and transchromosomal non-human animals, e.g., mice,will exhibit immunoglobulin production with a significant repertoire,ideally substantially similar to that of a human after adjusting forvolume.

The repertoire will ideally approximate that shown in a human whenadjusted for volume, usually with a diversity at least about 10% asgreat, preferably 25 to 50% or more. Generally, at least about athousand different immunoglobulins (ideally IgG), preferably 10⁴ to 10⁶or more, will be produced, depending on the number of different V, J andD regions introduced into the mouse genome and driven by the additionaldiversity generated by V(-D-)J gene segment rearrangements and randomnucleotide additions at the joining regions. Typically, theimmunoglobulins will exhibit an affinity (K_(D)) for preselectedantigens of about 10⁻⁸ M or less, 10⁻⁹ M or less, or 10⁻¹⁰ M or evenlower.

Transgenic and transchromosomal non-human animals, e.g., mice, asdescribed above can be immunized with, for example, cells producingIL-8. Alternatively, the transgenic animals can be immunized with DNAencoding human IL-8. The animals will then produce B cells which undergoclass-switching via switch recombination (cis-switching) and expressimmunoglobulins reactive with IL-8. The immunoglobulins will be humanantibodies (also referred to as “human sequence antibodies”), whereinthe heavy and light chain polypeptides are encoded by human transgenesequences, which may include sequences derived by somatic mutation and Vregion recombinatorial joints, as well as germline-encoded sequences;these human antibodies can be referred to as being substantiallyidentical to a polypeptide sequence encoded by human V_(L) and J_(L) orV_(H), D_(H) and J_(H) gene segments, even though other non-germlinesequences may be present as a result of somatic mutation anddifferential V-J and V-D-J recombination joints. The variable regions ofeach antibody chain are typically at least 80% similar to human germlineV, and J gene segments, and, in the case of heavy chains, human germlineV, D, and J gene segments; frequently at least 85% similar to humangermline sequences present on the transgene; often 90 or 95% or moresimilar to human germline sequences present on the transgene. However,since non-germline sequences are introduced by somatic mutation and VJand VDJ joining, the human sequence antibodies will frequently have somevariable region sequences which are not encoded by human V, D, or J genesegments as found in the human transgene(s) in the germline of the mice.Typically, such non-germline sequences (or individual nucleotidepositions) will cluster in or near CDRs, or in regions where somaticmutations are known to cluster.

Another aspect of the invention includes B cells derived from transgenicor transchromosomal non-human animals as described herein. The B cellscan be used to generate hybridomas expressing human monoclonalantibodies which bind with high affinity to human IL-8. Thus, in anotherembodiment, the invention provides a hybridoma which produces a humanantibody having an affinity (K_(D)) of about 10⁻⁸M or less, 10⁻⁹ M orless, 10⁻¹⁰ M or less when determined by surface plasmon resonance (SPR)technology in a BIACORE 3000 instrument using recombinant human IL-8 asthe analyte and the antibody as the ligand, or when determined byscatchard analysis of IL-8 expressing cells using a radio-activelylabeled monoclonal antibody, or by determination of the half-maximalbinding concentration using FACS analysis.

Herein the monoclonal antibody comprises a human sequence light chaincomposed of (1) a light chain variable region having a polypeptidesequence which is substantially identical to a polypeptide sequenceencoded by a human V_(L) gene segment and a human J_(L) segment, and (2)a light chain constant region encoded by a human C_(L) gene segment; and

a human sequence heavy chain composed of a (1) a heavy chain variableregion having a polypeptide sequence which is substantially identical toa polypeptide sequence encoded by a human V_(H) gene segment, a Dregion, and a human J_(H) segment, and (2) a constant region encoded bya human C_(H) gene segment.

The development of high affinity human monoclonal antibodies againstIL-8 can be facilitated by a method for expanding the repertoire ofhuman variable region gene segments in a transgenic non-human animalhaving a genome comprising an integrated human immunoglobulin transgene,said method comprising introducing into the genome a V gene transgenecomprising V region gene segments which are not present in saidintegrated human immunoglobulin transgene. Often, the V region transgeneis a yeast artificial chromosome (YAC) comprising a portion of a humanV_(H) or V_(L) (V_(K)) gene segment array, as may naturally occur in ahuman genome or as may be spliced together separately by recombinantmethods, which may include out-of-order or omitted V gene segments.Often at least five or more functional V gene segments are contained onthe YAC. In this variation, it is possible to make a transgenic animalproduced by the V repertoire expansion method, wherein the animalexpresses an immunoglobulin chain comprising a variable region sequenceencoded by a V region gene segment present on the V region transgene anda C region encoded on the human Ig transgene. By means of the Vrepertoire expansion method, transgenic animals having at least 5distinct V genes can be generated; as can animals containing at leastabout 24 V genes or more. Some V gene segments may be non-functional(e.g., pseudogenes and the like); these segments may be retained or maybe selectively deleted by recombinant methods available to the skilledartisan, if desired.

Once the mouse germline has been engineered to contain a functional YAChaving an expanded V segment repertoire, substantially not present inthe human Ig transgene containing the J and C gene segments, the traitcan be propagated and bred into other genetic backgrounds, includingbackgrounds where the functional YAC having an expanded V segmentrepertoire is bred into a non-human animal germline having a differenthuman Ig transgene. Multiple functional YACs having an expanded Vsegment repertoire may be bred into a germline to work with a human Igtransgene (or multiple human Ig transgenes). Although referred to hereinas YAC transgenes, such transgenes when integrated into the genome maysubstantially lack yeast sequences, such as sequences required forautonomous replication in yeast; such sequences may optionally beremoved by genetic engineering (e.g., restriction digestion andpulsed-field gel electrophoresis or other suitable method) afterreplication in yeast is no longer necessary (i.e., prior to introductioninto a mouse ES cell or mouse prozygote). Methods of propagating thetrait of human sequence immunoglobulin expression, include breeding atransgenic animal having the human Ig transgene(s), and optionally alsohaving a functional YAC having an expanded V segment repertoire. BothV_(H) and V_(L) gene segments may be present on the YAC. The transgenicanimal may be bred into any background desired by the practitioner,including backgrounds harboring other human transgenes, including humanIg transgenes and/or transgenes encoding other human lymphocyteproteins. The invention also provides a high affinity human sequenceimmunoglobulin produced by a transgenic mouse having an expanded Vregion repertoire YAC transgene. Although the foregoing describes apreferred embodiment of the transgenic animal of the invention, otherembodiments are contemplated which have been classified in threecategories:

I. Transgenic animals containing an unrearranged heavy and rearrangedlight chain immunoglobulin transgene;

II. Transgenic animals containing an unrearranged heavy and unrearrangedlight chain immunoglobulin transgene; and

III. Transgenic animal containing rearranged heavy and an unrearrangedlight chain immunoglobulin transgene; Of these categories of transgenicanimal, the preferred order of preference is as follows II>I>III wherethe endogenous light chain genes (or at least the κ gene) have beenknocked out by homologous recombination (or other method) and I>II>IIIwhere the endogenous light chain genes have not been knocked out andmust be dominated by allelic exclusion.

III. Pharmaceutical Compositions

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing at least one human monoclonalantibody of the present invention, formulated together with apharmaceutically acceptable carrier. In one embodiment, the compositionincludes a combination of multiple (e.g., two or more) isolated humanantibodies of the invention. Preferably, each of the antibodies of thecomposition binds to a distinct, pre-selected epitope of IL-8.

Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a composition of the present inventionwith at least one agent for treating inflammatory or hyperproliferativeskin disorders, at least one anti-inflammatory agent, at least oneimmunosuppressive agent, or at least one chemotherapeutic agent.

In one embodiment, such therapeutic agents include one or more agentsfor inflammatory or hyperproliferative skin disorders, such as topicalmedications, including coal tar, A vitamin, anthralin, calcipotrien,tarazotene, and corticosteroids, oral or injected medications, such ascorticosteroids, methotrexate, retinoids, e.g., acitretin, cyclosporine,etanercept, alefacept, efalizumab, 6-thioguanine, mycophenolate mofetil,tacrolimus (FK-506), and hydroxyurea. Other examples are CTLA4Ig andinfliximab. Other treatments may include exposure to sunlight orphototherapy, including UVB (broad-band and narrow-band ultraviolet B),UVA (ultraviolet A) and PUVA (psoralen methoxalen plus ultraviolet A).

In a further embodiment, the compositions of the invention areadministered in conjunction with two or more of the above therapies,such as methotrexate+phototherapy (PUVA or UVA); methotrexate+acitretin;acitretin+phototherapy (PUVA or UVA);methotrexate+acitretin+phototherapy (PUVA or UVB);hydroxyurea+phototherapy (PUVA or UVB); hydroxyurea+acitretin;cyclosporine+methotrexate; or calcipotrien+phototherapy (UVB).

In another embodiment such therapeutic agents include one or moreanti-inflammatory agents, such as a steroidal drug or a NSAID(nonsteroidal anti-inflammatory drug). Preferred agents include, forexample, aspirin and other salicylates, Cox-2 inhibitors, such asrofecoxib and celecoxib, NSAIDs such as ibuprofen, fenoprofen, naproxen,sulindac, diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone,etodolac, oxaprozin, and indomethacin.

In another embodiment, such therapeutic agents include one or moreDMARDs (disease modifying antirheumatic drugs), such as methotrexate,hydroxy-chloroquine, sulfasalazine, pyrimidine synthesis inhibitors,e.g., leflunomide, IL-1 receptor blocking agents, e.g., anakinra, andTNF-α blocking agents, e.g., etanercept, infliximab, and adalimumab.Further representatives are IL-10, anti-IL-15 antibodies, solubleIL-15R, and anti-CD20 antibodies.

In another embodiment, such therapeutic agents include one or moreimmunosuppressive agents, such as cyclosporine, azathioprine,mycophenolic acid, mycophenolate mofetil, corticosteroids, such asprednisone, methotrexate, gold salts, sulfasalazine, antimalarials,brequinar, leflunomide, mizoribine, 15-deoxyspergualine,6-mercaptopurine, cyclophosphamide, rapamycin, and tacrolimus (FK-506).

In another embodiment, the compositions of the invention areadministered in combination with two or more immunosuppressive agents,such as prednisone and cyclosporine; prednisone, cyclosporine andazathioprine; or prednisone, cyclosporine and mycophenolate mofetil.

In another embodiment, such therapeutic agents include one or morechemotherapeutics, such as doxorubicin, cisplatin, bleomycin, carmustin,cyclophosphamide, and chlorambucil.

In another embodiment, the present human monoclonal antibodies may beadministered in conjunction with radiotherapy.

In another embodiment, the human antibodies of the invention may beadministered in combination with one or more other antibodies, e.g., oneor more human antibodies such as, e.g., anti-CD4 antibodies, anti-EGFrantibodies, anti-CD20 antibodies, anti-IL15 antibodies, or anti-IL15Rantibodies.

In yet another embodiment, the human antibodies of the invention may beadministered in combination with one or more agents, that block orinterfere with the function of CC or CXC chemokine receptors, such asantibodies to CXCR1, CXCR2, CCR1, CCR2, or CCR5, or natural or syntheticmolecules that act as chemokine receptor antagonists.

In still another embodiment, the human antibodies of the invention maybe administered in combination with one or more agents, that block thefunction of chemokine ligands, such as antibodies to MIP-1α, MIP-1β,RANTES, MCP-1, MCP-2, MCP-3 or MCP-4.

Furthermore, the human anti-IL-8 antibodies of the present invention canbe derivatized, linked to or co-expressed with other bindingspecificities. In a particular embodiment, the invention provides abispecific or multispecific molecule comprising at least one firstbinding specificity for IL-8 (e.g., a human anti-IL-8 antibody ormimetic thereof), and a second binding specificity for a human effectorcell, such as a binding specificity for an Fc receptor (e.g., a humanFcγ receptor, such as FcγRI, or a human Fcα receptor) or a T cellreceptor, e.g., CD3.

Accordingly, the present invention includes bispecific and multispecificmolecules that bind to both human IL-8 and to an Fc receptor or a T cellreceptor, e.g., CD3. Examples of Fc receptors are, e.g., a human IgGreceptor, e.g., an Fc-gamma receptor (FcγR), such as FcγRI (CD64),FcγRII (CD32), and FcγRIII (CD16). Other Fc receptors, such as human IgAreceptors (e.g., FcαRT), also can be targeted. The Fc receptor ispreferably located on the surface of an effector cell, e.g., a monocyte,macrophage or an activated mononuclear cell. In a preferred embodiment,the bispecific and multispecific molecules bind to an Fc receptor at asite which is distinct from the immunoglobulin Fc (e.g., IgG or IgA)binding site of the receptor. Therefore, the binding of the bispecificand multispecific molecules is not blocked by physiological levels ofimmunoglobulins.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity and absorption delaying agents, and the like thatare physiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion).

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, S. M., et al.(1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acidaddition salts and base addition salts. Acid addition salts includethose derived from nontoxic inorganic acids, such as hydrochloric,nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous acidsand the like, as well as from nontoxic organic acids such as aliphaticmono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acidsand the like. Base addition salts include those derived from alkalineearth metals, such as sodium, potassium, magnesium, calcium and thelike, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A composition of the present invention can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results. The active compounds can be prepared withcarriers that will protect the compound against rapid release, such as acontrolled release formulation, including implants, transdermal patches,and microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

To administer a compound of the invention by certain routes ofadministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the compound may be administered to a subject in anappropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al. (1984) J. Neuroimmunol. 7:27).

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonicity agents, for example, sugars, polyalcohols such asglycerol, mannitol, sorbitol, or sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

In one embodiment the human monoclonal antibodies of the invention areadministered in crystalline form by subcutaneous injection, cf. Yang etal. (2003) PNAS, 100(12):6934-6939.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Examples of pharmaceutically-acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

For the therapeutic compositions, formulations of the present inventioninclude those suitable for oral, nasal, topical (including buccal andsublingual), rectal, vaginal and/or parenteral administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods known in the art of pharmacy. The amount ofactive ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the subject beingtreated, and the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compositionwhich produces a therapeutic effect. Generally, out of 100%, this amountwill range from about 0.01% to about 99% of active ingredient,preferably from about 0.1% to about 70%, most preferably from about 1%to about 30%.

Formulations of the present invention which are suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate. Dosage forms for the topical or transdermaladministration of compositions of this invention include powders,sprays, ointments, pastes, creams, lotions, gels, solutions, patches andinhalants. The active compound may be mixed under sterile conditionswith a pharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection or infusion, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol, sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

When the compounds of the present invention are administered aspharmaceuticals, to humans and animals, they can be given alone or as apharmaceutical composition containing, for example, 0.01 to 99.5% (morepreferably, 0.1 to 90%) of active ingredient in combination with apharmaceutically acceptable carrier.

Regardless of the route of administration selected, the compounds of thepresent invention, which may be used in a suitable hydrated form, and/orthe pharmaceutical compositions of the present invention, are formulatedinto pharmaceutically acceptable dosage forms by conventional methodsknown to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitabledaily dose of a compositions of the invention will be that amount of thecompound which is the lowest dose effective to produce a therapeuticeffect. Such an effective dose will generally depend upon the factorsdescribed above. It is preferred that administration be intravenous,intramuscular, intraperitoneal, or subcutaneous, preferably administeredproximal to the site of the target. If desired, the effective daily doseof a therapeutic composition may be administered as two, three, four,five, six or more sub-doses administered separately at appropriateintervals throughout the day, optionally, in unit dosage forms. While itis possible for a compound of the present invention to be administeredalone, it is preferable to administer the compound as a pharmaceuticalformulation (composition). The dosage can be determined or adjusted bymeasuring the amount of circulating monoclonal anti-IL-8 antibodies atdifferent time points following administration in a biological sample bymaking use of anti-idiotypic antibodies targeting the anti-IL-8antibodies or by using other specific methods to detect the anti-IL8antibodies for instance by an ELISA assay using IL-8 as coating.

In one embodiment, the human monoclonal antibodies according to theinvention may be administered by infusion in a dosage of 0.15 to 8mg/kg, e.g., 0.15 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, 4mg/kg, or 8 mg/kg, on day 0 followed by 2 to 8 administrations once aweek, such as 4 administrations once a week starting at day 28. Theadministration may be performed by continuous infusion over a period of24 hours or over a period of more than 24 hours, in order to reducetoxic side effects.

In yet another embodiment, the human monoclonal antibodies areadministered by maintenance therapy, such as, e.g., once a week, onceevery second week or once a month for a period of 6 months or more.

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a preferred embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicaments through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system. Many othersuch implants, delivery systems, and modules are known to those skilledin the art.

In certain embodiments, the human monoclonal antibodies of the inventioncan be formulated to ensure proper distribution in vivo. For example,the blood-brain barrier (BBB) excludes many highly hydrophiliccompounds. To ensure that the therapeutic compounds of the invention cancross the BBB (if desired), they can be formulated, for example, inliposomes. For methods of manufacturing liposomes, see, e.g., U.S. Pat.Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may comprise oneor more moieties which are selectively transported into specific cellsor organs, thus enhancing targeted drug delivery (see, e.g., V. V.Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moietiesinclude folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low etal.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun.153:1038); antibodies (P. G. Bloeman et al. (1995) FEBS Lett. 357:140;M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactantprotein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134),different species of which may comprise the formulations of theinventions, as well as components of the invented molecules; p 120(Schreier et al. (1994) J. Biol. Chem. 269:9090); see also K. Keinanen;M. L. Laukkanen (1994) FEBS Lett. 346:123; J. J. Killion; I. J. Fidler(1994) Immunomethods 4:273. In one embodiment of the invention, thetherapeutic compounds of the invention are formulated in liposomes; in amore preferred embodiment, the liposomes include a targeting moiety. Ina most preferred embodiment, the therapeutic compounds in the liposomesare delivered by bolus injection to a site proximal to the desired area,e.g., the site of inflammation or infection, or the site of a tumor. Thecomposition must be fluid to the extent that easy syringability exists.It must be stable under the conditions of manufacture and storage andmust be preserved against the contaminating action of microorganismssuch as bacteria and fungi.

The efficient dosages and the dosage regimens for the human monoclonalantibodies of the invention depend on the disease or condition to betreated and can be determined by the persons skilled in the art.

A “therapeutically effective dosage” for PPP preferably will result in areduction in the overall PPP evaluation comparing impression ofimprovement after drug treatment with pretreatment condition. This can,e.g., be evaluated by the reduction in the number of fresh pustules, aPASI like score adapted to PPP denoted PPPASI. Preferably, the treatmentwill result in a PPPASI50, more preferably a PPPASI75, and even morepreferably a PPPASI90.

A “therapeutically effective dosage” for psoriasis preferably willresult in a PASI50, more preferably a PASI75, and even more preferably aPASI90 in the patients or a reduction in the overall psoriasisevaluation comparing impression of improvement after drug treatment withpretreatment condition. PASI (Psoriasis Area and Severity Index) is ascore system used for evaluation of the area and severity of thedisease. PASI50 is defined as ≥50% improvement of the score. In the sameway, PASI75 and PASI90 are defined as ≥75% and ≥90% improvement of thescore, respectively.

A “therapeutically effective dosage” for rheumatoid arthritis preferablywill result in an ACR20 Preliminary Definition of Improvement in thepatients, more preferred in an ACR50 Preliminary Definition ofImprovement and even more preferred in an ACR70 Preliminary Definitionof Improvement.

ACR20 Preliminary Definition of Improvement is defined as: ≥20%improvement in: Tender Joint Count (TJC) and Swollen Joint Count (SJC)and ≥20% improvement in 3 of following 5 assessments: Patient PainAssessment (VAS), Patient Global assessment (VAS), Physician GlobalAssessment (VAS), Patient Self-Assessed Disability (HAQ), and AcutePhase Reactant (CRP or ESR).

ACR50 and ACR70 are defined in the same way with ≥50% and ≥70%improvements, respectively. For further details see Felson et al. inAmerican College of Rheumatology Preliminary Definition of Improvementin Rheumatoid Arthritis; Arthritis Rheumatism (1995) 38:727-735.

Alternatively, a therapeutically effective dosage for rheumatoidarthritis can be measured by DAS (disease activity score), includingDAS28 and more preferably DAS56, as defined by EULAR.

A “therapeutically effective dosage” for tumor therapy can be measuredby objective tumor responses which can either be complete or partial. Acomplete response (CR) is defined as no clinical, radiological or otherevidence of disease. A partial response (PR) results from a reduction inaggregate tumor size of greater than 50%. Median time to progression isa measure that characterizes the durability of the objective tumorresponse.

A “therapeutically effective dosage” for tumor therapy can also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer can be evaluated in an animalmodel system predictive of efficacy in human tumors. A therapeuticallyeffective amount of a therapeutic compound can decrease tumor size, orotherwise ameliorate symptoms in a subject. One of ordinary skill in theart would be able to determine such amounts based on such factors as thesubject's size, the severity of the subject's symptoms, and theparticular composition or route of administration selected.

IV. Uses and Methods of the Invention

The human antibodies and antibody compositions of the present inventionhave numerous in vivo and in vitro therapeutic and diagnostic utilitiesinvolving the treatment and diagnosis of IL-8 mediated disorders ordisorders involving IL-8 activity. These molecules can be administeredto human subjects, e.g., in vivo, or to cells in culture, e.g., in vitroor ex vivo, to treat, prevent and to diagnose a variety of disorders. Asused herein, the term “subject” is intended to include human andnon-human animals). Preferred subjects include human patients havingdisorders caused by or associated with IL-8 activity.

More particularly, the human antibodies and derivatives thereof are usedto inhibit IL-8 induced activities associated with certain disorders,e.g., proinflammatory activity, chemotactic activity, and angiogenesis.Other IL-8 induced activities which are inhibited by the antibodies ofthe present invention include inhibiting IL-8 induced increasedexpression of CD11b (Mac-1) and inhibiting IL-8 induced decreasedexpression of L-selectin. By contacting the antibody with IL-8 (e.g., byadministering the antibody to a subject), the ability of IL-8 to bind toits receptors and to subsequently induce such activities is inhibitedand, thus, the associated disorder is treated. Preferred antibodies bindto epitopes which are specific to IL-8 and, thus, advantageously inhibitIL-8 induced activities, but do not interfere with the activity ofstructurally related chemokines, such as GRO-α, GRO-β, IP-10 and NAP-2.

In one embodiment, the human antibodies of the invention can be used inmethods for treating inflammatory or hyperproliferative skin disorders,such as PPP, psoriasis, including plaque psoriasis and guttate typepsoriasis, bullous skin diseases, such as bullous pemphigoid, contactdermatitis, eczema, erythematosus, and atopic dermatitis.

In another embodiment, the human antibodies of the invention can be usedin methods for treating immune, autoimmune, inflammatory or infectiousdiseases, such as psoriatic arthritis, systemic scleroderma andsclerosis, inflammatory bowel disease (IBD), Crohn's disease, ulcerativecolitis, acute lung injury, such as acute respiratory distress syndromeor adult respiratory distress syndrome, meningitis, encephalitis,uveitis, multiple myeloma, glomerulonephritis, nephritis, asthma,atherosclerosis, leukocyte adhesion deficiency, multiple sclerosis,Raynaud's syndrome, Sjögren's syndrome, juvenile onset diabetes,Reiter's disease, Behcet's disease, immune complex nephritis, IgAnephropathy, IgM polyneuropathies, immune-mediated thrombocytopenias,such as acute idiopathic thrombocytopenic purpura and chronic idiopathicthrombocytopenic purpura, hemolytic anemia, myasthenia gravis, lupusnephritis, lupus erythematosus, rheumatoid arthritis (RA), ankylosingspondylitis, pemphigus, Graves' disease, Hashimoto's thyroiditis, smallvessel vasculitides, such as Wegener's granulomatosis, Omen's syndrome,chronic renal failure, autoimmune thyroid disease, acute infectiousmononucleosis, HIV, herpes virus associated diseases, human virusinfections, such as common cold as caused by human rhinovirus,coronavirus, other enterovirus, herpes virus, influenza virus,parainfluenza virus, respiratory syncytial virus or adenovirusinfection, bacteria pneumonia, wounds, sepsis, cerebral stroke/cerebraledema, ischaemia-reperfusion injury and hepatitis C.

In one embodiment, the human monoclonal antibodies can be used for thetreatment of ischaemia-reperfusion injury after thrombolysis,cardiopulmonary bypass, percutaneous coronary intervention (PCI),coronary artery bypass, or cardiac transplantation.

In yet another embodiment, the human antibodies of the invention can beused for treatment of alcoholic hepatitis and acute pancreatitis.

In yet a further embodiment, the human antibodies of the invention canbe used in methods for treating diseases involving IL-8 mediatedangiogenesis, such as tumors and cancers, e.g., melanoma, thyroidcarcinoma, transitional cell carcinoma, trichilemmona, squamous cellcarcinoma and breast cancer.

In another embodiment, the human antibodies of the invention can be usedfor treating diseases wherein blocking of granulocyte migration isbeneficial, e.g., in diseases affecting the central nervous system, suchas isolated cerebral angiitis;

diseases affecting the peripheral nervous system, such as mononeuritismultiplex;

cardiovascular disorders, such as acute myocardial infarction,myocarditis, pericarditis, and Liebman-Sachs endocarditis;

pulmonary disorders, such as chronic obstructive pulmonary disease(COPD), alveolitis, obliterating bronchiolitis, cystic fibrosis,allergic aspergillosis, and Löfflers syndrome;

hepatic disorders, such as sclerosing cholangiolitis;

urogenital disorders, such as chronic cyctitis;

renal disorders, such as tubulo-interstial nephritis;

infectious diseases, such as severe acute respiratory syndrome (SARS);

rheumatic disorders, such as large vessel vasculitides (including giantcell arteritis, polymyalgia rheumatica, and Takayasu arteritis),medium-sized vessel vasculitides (including polyarteritis nodosa,localized polyarteritis nodosa, and Kawasaki disease), small vesselvasculitides (including Churg-Strauss syndrome, microscopicpolyarteritis, cryoglobulinemic vasculitis, and leucocytoclasticangiitis), secondary vasculitides (including rheumatoid vasculitis, andvasculitis associated with systemic lupus erythematosus or Sjögren'ssyndrome), isolated sacroileitis, the SAPHO syndrome, and disciitis(including postoperative disciitis);

endocrine disorders, such as subacute thyroiditis;

skin disorders, such as cicatricial pemphigoid, dermatitisherpetiformis, subcorneal pustular dermatosis, epidermolysis bullosaacquisita, rosacea, acute febrile dermatosis, granuloma annulare(including Sweet's syndrome), pyoderma gangraenosum, and acne (includingacne conglobata);

connective tissue disorders, such as sarcoidosis, relapsingpolychondritis, familial Mediterranean fever, panniculitis, erythemanodosum, Weber-Christian's disease, and retroperitoneal fibrosis.

In another embodiment, the human antibodies of the invention are usedfor treating diseases wherein interfering with interactions between IL-8and osteoclasts is beneficial, such as osteoporosis, and osteolyticmetastases.

In another embodiment, the human antibodies of the invention are usedfor treating disease wherein interfering with interactions between IL-8and tumor cells is beneficial, such as gastric cancer, colorectalcancer, and urine bladder cancer.

The methods involve administering to a subject an antibody compositionof the present invention in an amount effective to treat or prevent thedisorder. The antibody composition can be administered alone or alongwith one or more further therapeutic agents, such as one or more agentsselected from agents for treating inflammatory or hyperproliferativeskin disorders, anti-inflammatory agents, immunosuppressive agents, andchemotherapeutic agents, which act in conjunction with orsynergistically with the antibody composition to treat or prevent theIL-8 mediated disease.

Suitable routes of administering the antibody compositions of theinvention in vivo and in vitro are well known in the art and can beselected by those of ordinary skill. For example, the antibodycompositions can be administered by injection or infusion (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject, the concentrationand/or formulation of the antibody composition, and the disease beingtreated.

As previously described, human anti-IL-8 antibodies of the invention canbe co-administered with one or other more therapeutic agents. Theantibody can be administered before, after or concurrently with theagent.

Also within the scope of the present invention are kits comprising theantibody compositions of the invention and instructions for use. The kitcan further contain one ore more additional agents, such as animmunosuppressive agent, or one or more additional human antibodies ofthe invention (e.g., a human antibody having a complementary activitywhich binds to an epitope in the IL-8 antigen distinct from the firsthuman antibody).

Accordingly, patients treated with antibody compositions of theinvention can be additionally administered (prior to, simultaneouslywith, or following administration of a human antibody of the invention)with another therapeutic agent, such as an anti-inflammatory agent,which enhances or augments the therapeutic effect of the humanantibodies.

In yet another embodiment, the invention provides methods for diagnosingdiseases associated with IL-8 by detection ex vivo or in vitro of IL-8in a sample, e.g., a tissue sample, a body fluid sample or a cellsample. This can be achieved, for example, by contacting a sample to betested, optionally along with a control sample, with the human antibodyunder conditions that allow for formation of a complex between theantibody and IL-8. Complex formation can then be detected (e.g., usingan ELISA). When using a control sample along with the test sample,complex can be detected in both samples and any statisticallysignificant difference in the formation of complexes between the samplesis indicative of the presence of IL-8 in the test sample.

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting.

EXAMPLES Example 1 Production of Human Monoclonal Antibodies (HuMabs)Against IL-8

Human monoclonal antibodies against human IL-8 (72 amino acid form) wereproduced as follows in transgenic mice carrying human immunoglobulintransgenes.

Antigen:

Recombinant human IL-8 antigen (rhIL-8) was prepared using standardrecombinant DNA techniques and provided at a protein concentration of0.713 mg/mL in PBS. The soluble antigen was stored at −80° C. until use.Soluble IL-8 was mixed with Complete Freunds Adjuvant (CF) (Sigma F5881)for the first immunization. Thereafter, the antigen was mixed withIncomplete Freunds Adjuvant (IF) (Sigma F5506). Twenty-five microgramsof recombinant IL-8 in 100 μL PBS was mixed 1:1 with the adjuvant usingan emulsifying needle. Mice were injected with 0.2 mL prepared antigeninto the intraperitoneal cavity.

Transgenic Mice:

Mice were housed in filter cages and were evaluated to be in goodphysical condition on the dates of immunization and bleeds, and on theday of the fusion.

The mouse that produced monoclonal antibody (mAb) 10F8 was a male, ID#81645 of the (CMD)++; (HCo7) 19952+; (JKD)++; (KCoS) 9272+ genotype.Individual transgene designations are in parentheses, followed by linenumbers for randomly integrated transgenes. The symbols ++ and +indicate homozygous or hemizygous. However, because the mice areroutinely screened using a PCR-based assay that does not allowdistinction between heterozygosity and homozygosity for the randomlyintegrated human Ig transgenes, a + designation may be given to micethat are actually homozygous for these elements.

The HCo7 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al. (1993) EMBO J. 12: 821-830),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424), a KCoS human kappa light chain transgene (asdescribed in Fishwild et al. (1996) Nature Biotechnology 14:845-851),and a HCo7 human heavy chain transgene (as described in U.S. Pat. No.5,770,429).

The HCo12 mice have a JKD disruption in their endogenous light chain(kappa) genes (as described in Chen et al. (1993) EMBO J. 12: 821-830),a CMD disruption in their endogenous heavy chain genes (as described inExample 1 of WO 01/14424 by Korman et al.), a KCoS human kappa lightchain transgene (as described in Fishwild et al. (1996) NatureBiotechnology 14:845-851), and a HCo12 human heavy chain transgene (asdescribed in Example 2 of WO 01/14424 by Korman et al.).

Immunization Procedure:

The immunization schedule used for the mice is listed in Table 1 below.Splenocytes of ten mice from HCo7 and HCo12 genotypes immunized withrIL-8 were fused on Day 101.

TABLE 1 Dates Procedure Day 0 Immunization 25 μg IL-8 ip CF Day 15Immunization 25 μg IL-8 ip IF Day 28 Immunization 25 μg IL-8 ip IF Day38 Titer Day 50 Immunization 25 μg IL-8 ip IF Day 59 Titer Day 63Immunization 25 μg IL-8 ip IF Day 73 Titer Day −3 and −2 prior to fusionImmunization 25 μug IL-8 iv Day 101 Fusion

Hybridoma Preparation:

The P3 X63 ag8.653 myeloma cell line (ATCC CRL 1580, lot F-15183) wasused for the fusions. The original ATCC vial was thawed and expanded inculture. A seed stock of frozen vials was prepared from this expansion.Cells were maintained in culture for 3-6 months, passed twice a week.P388D1 cell line (ATCC TIB-63 FL) was expanded to 200 mL and exhausted.The supernatant was spun down and filtered and used as a media additionknown as conditioned media. This cell line was passed for 3-6 months andthen a new vial was thawed.

High Glucose DMEM (Mediatech, Cellgro #10013245) containing 5% FBS, andPenicillin-Streptomycin (Cellgro #30004030) was used to culture themyeloma and P388D1 cells. Additional media supplements were added to thehybridoma growth media, which included: 3% Origen-Hybridoma CloningFactor (Igen, 36335), 10% P388D1 conditioned media (8/10/99 DH), 10% FBS(Hyclone, SH30071 lot #AGH6843), L-glutamine (Gibco #1016483) 0.1%gentamycin (Gibco #1020070), 2-mercaptoethanol (Gibco #1019091), HAT((Sigma, H0262) 1.0×10⁴ M hypoxanthine, 4.0×10⁻⁷ M aminopterin, 1.6×10⁻⁵M thymidine), or HT ((Sigma, H0137) 1.0×10⁻⁴ M hypoxanthine, 1.6×10⁻⁵ Mthymidine).

The spleen from mouse #18645 was normal in size and yielded 1.8×10⁸viable cells. 10 (96-well) plates were dispensed at 200 μL/well. Thesplenocytes were then fused and an initial ELISA screen for human IgG,κantibodies was performed 10-12 days post fusion.

Human IgG,κ positive wells were then screened on soluble IL-8 coatedELISA plates. Antigen positive hybridomas were then transferred to24-well plates, and eventually to tissue culture flasks. IL-8 specifichybridomas were subcloned by limiting dilution to assure monoclonality.Antigen positive hybridomas were preserved at several stages in thedevelopment process by freezing cells in DMEM supplemented with 50% FBSplus 10% DMSO (Sigma, D2650).

The titers for mouse #18645 were as shown below in Table 2. The titersare Huγ antigen specific. Titer values are defined as the reciprocal ofthe highest dilution resulting in an OD equal to twice that ofbackground.

TABLE 2 Date Titer Day 0 200 Day 21 1600 Day 35 1600-3200

The fusion was screened for Huγ antigen reactivity by ELISA. Followingthe screen for antigen (ELISA based), two possible antigen specifichybridomas were identified from the fusion. These two hybridomas, alongwith six other hybridomas from prior fusions, were evaluated fortherapeutic potential. These lines were subcloned and exhaustedsupernatants were purified over Protein-A. Determinations of K_(D)'swere done using BIAcore. When compared to the control mAb, hybridoma10F8 was identified to have very high affinity and was selected forfurther characterization.

Example 2 Determination of the V_(H) and V_(L) Regions of the Antibodies

Cell Culture

HuMab 10F8 hybridoma cell line was cultured in Dulbecco's Modified EagleMedium (DMEM, Gibco BRL) supplemented with 10% FCS, 2 mM L-glutamine,100 IU/mL penicillin, 100 μg/mL streptomycin (pen/strep) (all derivedfrom Gibco BRL, Life Technologies, Paisley, U.K.) and 1 mM sodiumpyruvate. Cells were kept at 37° C. in a humidified atmospherecontaining 5% CO₂.

RNA Preparation:

PolyA+ mRNA was prepared from 2×10⁶ HuMab-IL-8 (10F8) cells using theMicro-Fast Track Kit (Invitrogen, Carlsbad, Calif., U.S.A.), followingthe manufacturer's protocol.

cDNA Preparation:

Complementary DNA (cDNA) of RNA from HuMab-IL-8 (10F8) cells wasprepared from ¼ of the mRNA obtained, using the cDNA Cycle Kit(Invitrogen, Carlsbad, Calif., U.S.A.), following the manufacturer'sprotocol.

V_(H) and V_(L) regions were amplified using the following primers:

V_(H) FR1 5′ primers: AB62 (SEQ ID NO: 25) CAg gTK CAg CTg gTg CAg TC AB63 (SEQ ID NO: 26) SAg gTg CAg CTg KTg gAg TC  AB65 (SEQ ID NO: 27)gAg gTg CAg CTg gTg CAg TC  V_(H) leader 5′ primers: AB85(SEQ ID NO: 28) ATg gAC Tgg ACC Tgg AgC ATC  AB86 (SEQ ID NO: 29)ATg gAA TTg ggg CTg AgC Tg  AB87 (SEQ ID NO: 30)ATg gAg TTT ggR CTg AgC Tg  AB88 (SEQ ID NO: 31)ATg AAA CAC CTg Tgg TTC TTC  AB89 (SEQ ID NO: 32)ATg ggg TCA ACC gCC ATC CT  V_(H) 3′ primer: AB90 (SEQ ID NO: 33)TgC CAg ggg gAA gAC CgA Tgg  V_(K) FR1 5′ primers: AB8 (SEQ ID NO: 34)RAC ATC CAg ATg AYC CAg TC  AB9 (SEQ ID NO: 35)gYC ATC YRg ATg ACC CAg TC  AB10 (SEQ ID NO: 36)gAT ATT gTg ATg ACC CAg AC  AB11 (SEQ ID NO: 37)gAA ATT gTg TTg ACR CAg TC  AB12 (SEQ ID NO: 38)gAA ATW gTR ATg ACA CAg TC  AB13 (SEQ ID NO: 39)gAT gTT gTg ATg ACA CAG TC  AB14 (SEQ ID NO: 40)gAA ATT gTg CTg ACT CAg TC  V_(K) leader 5′ primers: AB123(SEQ ID NO: 41) CCC gCT Cag CTC CTg ggg CTC CTg  AB124 (SEQ ID NO: 42)CCC TgC TCA gCT CCT ggg gCT gC  AB125 (SEQ ID NO: 43)CCC AgC gCA gCT TCT CTT CCT CCT gC  AB126 (SEQ ID NO: 44)ATg gAA CCA Tgg AAg CCC CAg CAC AgC  V_(K) 3′ primer: AB16(SEQ ID NO: 45) Cgg gAA gAT gAA gAC AgA Tg 

In the above primer sequences, K, S, R, Y and W have the followingmeanings:

K=G or T; S=C or G; R=A or G; Y=C or T; and W=A or T

PCR conditions used to amplify V_(H) and V_(L) regions for cloning:Polymerase chain reactions (PCR) were performed with cloned Pfupolymerase (Stratagene, La Jolla, Calif., U.S.A.) on a GeneAmp PCRSystem 9700 (Perkin Elmer Applied Biosystems, Foster City, Calif., USA).

PCR cycling protocol: 10 cycles 94° C. 2 min 94° C. 45 sec 65° C. 45sec, minus 1° C. per cycle 72° C. 1 min 20 cycles 94° C. 45 sec 55° C.45 sec 72° C. 1 min 72° C. 10 min cool down to 4° C.

Cloning of V_(H) and V_(L) in pCR-Blunt-Vector System:

After analysing the PCR products on an agarose gel, the products wereligated directly into the pCR-Blunt vector system (Invitrogen) accordingto the manufacturer's protocol. Three independently amplified V_(H) PCRproducts, and five independently amplified V_(L) PCR products, using FR1or leader primers, were cloned and sequenced.

After transformation into E. coli TOP 10 (Invitrogen), plasmid DNA fromcolonies was purified using the Qiaprep Spin miniprep kit (Qiagen,Valencia, Calif., U.S.A). Individual clones were screened for V_(H) orV_(L) PCR product insert by digestion with EcoRI (New England Biolabs,Beverly, Mass., U.S.A.) and analysis on an agarose gel.

Sequencing:

The V-regions were sequenced after cloning in the pCR-Blunt VectorSystem, using T7 and T3 primers, by ACGT, Inc., Northbrook, Ill., U.S.A.The sequences were analysed with the program DNAStar, SeqmanII. Thesequences were aligned to germline V-gene sequences in Vbase (See thewebsite of the MRC Centre for Protein Engineering (Cambridge, UK),mrc-cpe.cam.ac.uk/imt-doc/public/intro.htm).

The germline family for the V_(H)-region of 10F8 according to alignmentin Vbase: V_(H)3-33 (V_(H)3-subgroup), J_(H)4 (b) (J_(H)-segment). Nocomplementary regions for D_(H)-segment could be recognized by V-basesoftware, probably due to somatic hypermutations in the D-segment.

The germline family for the V_(L)-region of 10F8 according to alignmentin Vbase: V_(K) A-27 (V_(K)III-subgroup) and J_(K)3 (J_(K)-segment).

FIG. 1 shows the nucleotide sequences of the V_(L) and V_(H) regions of10F8.

FIGS. 2 and 4 show the alignment of the 10F8 V_(L) and V_(H) regionnucleotide sequences, respectively, with their corresponding germlinesequences. FIGS. 3 and 5 show the alignment of the 10F8 V_(L) and V_(H)region amino acid sequences, respectively, with their correspondinggermline-encoded sequences.

10F8: a human monoclonal IgG1,κ antibody with V_(H) and V_(L) amino acidsequences: SEQ ID NOs: 12 and 8, respectively.

Example 3 Expression of Recombinant HuMab 10F8

The cloned V_(L) and V_(H) regions from HuMab 10F8 were subcloned intothe expression cassettes of an immunoglobulin expression vector. The Vregions were inserted upstream of human kappa and gamma1 constantregions and encode full-length 10F8 heavy and light chains. The 10F8expression vector was transfected into Chinese hamster ovary (CHO) cellsand transfectoma cell lines expressing the recombinant antibody wereestablished. The affinity of recombinant 10F8 produced from CHO cellswas measured as being identical to the affinity of the hybridoma-derived10F8 antibody as assessed by kinetic analyses of plasmon surfaceresonance using a BIAcore.

Example 4 Binding of HuMab 10F8 (Fab and IgG) to IL-8

Purification of monoclonal antibody from culture supernatant: HuMab 10F8was purified by Protein-A affinity chromatography using the followingprocedure: (1) Loading Conditions: Supernatant was loaded on a 5 mLProtein-A column that was equilibrated with phosphate buffered saline(PBS); (2) Wash: PBS; (3) Elution: 0.1 M glycine with 150 mM NaCl, pH2.9. The eluate was neutralized with 1M Tris buffer (30 μl for every 2mL fraction). Each eluted fraction was run on gel before being pooled.Once the purity by coomassie staining was verified, fractions werepooled and dialyzed against 10 mM sodium phosphate buffer with 150 mMNaCl, pH 7.2.

Fab Fragment Preparation:

A Fab fragment preparation from HuMab 10F8 was performed according tokit instructions (Pierce Technical literature 44885). Five mg of thepurified IgG was used for this purpose. The isolated Fab product wasdialyzed against 10 mM sodium phosphate with 150 mM NaCl, pH 7.2 and itsprotein concentration was determined by BCA (Pierce) assay using BSA asa standard. The Fab was characterized for its purity and identity bySDS-PAGE.

Affinity Constants:

Affinity constants for 10F8 Fab were determined and compared withcorresponding values of 10F8 IgG1,κ. Whole IgG molecules lead torebinding effects during the dissociation phase of experimentalprocedures to determine the affinity constants, thus leading to anapparently lower dissociation rate constant and in turn, much higheraffinity constant. To eliminate these artifactual avidity effects, Fabmolecules were used in the place of IgG1,κ to determine the affinity andrate constants. A CM-5 chip was used to immobilize IL-8 via aminecoupling.

Using a BIAcore 3000, the association and rate constants based onsensograms (data not shown) of IgG1,κ and Fab at 25° C. and 37° C. aresummarized below.

Association and rate constants @ 25° C. IgG Fab k_(a) (×10⁵/M⁻¹ × sec⁻¹)2.31 1.01 k_(d) (×10⁻⁵ sec⁻¹) 0.21 1.96 K_(D) (×10⁻¹⁰M) 0.1 1.94Half-life (1n (2/k_off hrs)) 90.4 8.3

Association and rate constants @ 37° C. IgG Fab k_(a) (×10⁵/M⁻¹ × sec⁻¹)2.75 1.06 k_(d) (×10⁻⁵ sec⁻¹) 0.54 3.94 K_(D) (×10⁻¹⁰M) 0.2 3.72Half-life (1n (2/k_off hrs)) 38.2 4.9

Interaction of intact IgG1,κ with IL-8 yielded a dissociation rateconstant of 0.21×10⁻⁵ sec⁻¹, while the corresponding value for Fab was1.96×10⁻⁵ sec, indicating that rebinding and avidity effects influencethe dissociation rate constant of IgG1,κ (yielding a higher affinityconstant). These artifacts were eliminated with the use of Fab in theplace of intact IgG1,κ.

Analysis of the interaction at the physiological temperature of 37° C.,as compared to 25° C. (room temperature), showed that the rate constantsare further affected, leading to relatively lower affinity constants,for both IgG1,k and Fab. The affinity constant of the Fab at 37° C.corresponds to the true affinity of each binding site at physiologicaltemperature.

The half-life (i.e., the time taken for 50% of the complex todissociate), was reduced by 50% at 37° C. This is an approximation ofthe actual biological half-life at physiological temperatures.

Unless otherwise stated, the hybridoma derived 10F8 antibody has beenused in the following examples.

Example 5 Binding of HuMab 10F8 to Native IL-8 without Cross-Reactingwith Other CXC Chemokines

Possible cross-reactivity of HuMab 10F8 clone towards other chemokineswas evaluated by ELISA. Briefly, microtiter ELISA plates (Greiner,Germany) were coated overnight at room temperature (RT) with 1 μg/mL ofrecombinant human (rh) GRO-α, rh-GRO-β, rh-IP-10, rh-IL-8 72 aa form,which is monocyte derived (IL-8M or IL-8Mpepretech) or rh-IL-8 77 aaform (IL-8E), which is endothelial cell derived, 100 μl per well. Plateswere washed twice with PBST (phosphate buffered saline supplemented with0.05% v/v Tween-20 (Fischer Scientific, USA)) and wells were blockedwith 100 Owen PBSTC (PBST plus 2% v/v chicken serum) for 1 hour, RT.Thereafter, wells were incubated for 1 hour, RT under shaking conditionswith HuMab-IL-8 clone 10F8, 20 μg/mL 1:3 diluted in PBSTC. Subsequently,wells were washed thrice with PBST and incubated with eitherHRP-conjugated rabbit anti-mouse IgG F(ab′)₂ fragments (Jackson; diluted1:3000 in PBSTC) or HRP-conjugated rabbit anti-human IgG F(ab′)₂ (DAKO,Denmark; diluted 1:2000 in PBSTC) for the detection of mouse or humanantibodies, respectively. Plates were washed thrice with PBST and assayswere developed with freshly prepared ABTS solution (1 mg/mL) (ABTS:2,2′-azino bis (3-ethylbenzthiazoline-6-sulfonic acid; 2 tablets of 5 mgin 10 mL ABTS buffer, Boehringer Mannheim, Germany) for 30 minutes at RTin the dark. Absorbance was measured at 405 nm in an ELISA plate reader(Biotek Instruments Inc., Winochi, USA). The results shown in FIG. 6 arerepresentative out of three experiments performed. As it appears HuMab10F8 binds to both endothelial cell derived human IL-8 and to monocytederived human IL-8. However, it does not cross-react with the chemokinesGRO-α, GRO-β or IP-10.

Example 6 Inhibition of IL-8 Binding to IL-8R on Neutrophils

The ability of HuMab 10F8 to inhibit radiolabeled IL-8 binding to IL-8receptors (CXCR1 and CXCR2) on neutrophils was assessed as follows:

Neutrophils were enriched from heparinized whole blood from normalvolunteers. The blood was layered on Ficoll-hypaque and centrifuged at1500 rpm for 30 minutes. The mononuclear cell layer was removed, anderythrocytes were hypotonically lysed. The resulting neutrophils wereresuspended in PBS containing 0.1% BSA and 0.02% Na azide (0.1% PBA) andheld on ice. The IL-8 binding assay was performed as previouslydescribed (Yang et al, (1999) J. Leukoc. Biol. 66:401-410). Briefly, ina final volume of 150 μl, 4×10⁵ neutrophils were incubated from 1.5-3hours on ice with 0.25 nM [¹²⁵I] recombinant human IL-8 (Amersham LifeSciences, Piscataway, N.J.) along with varying concentrations of 10F8(hybridoma-derived), 10F8 (transfectoma-derived), mouse anti-human IL-8mAb 6712.111 R & D Systems, and control antibody. All incubations wereperformed in 96 well Multiscreen filter plates (Millipore, Bedford,Mass.). Plates were washed extensively with cold 0.1% PBA, and filterswere counted on a Wallac gamma counter. Results are shown in FIGS. 7Aand 7B, expressed as means of triplicate or quadruplicates. Theinhibition of binding is expressed as percentage of control antibody.

As shown in FIGS. 7A and 7B, HuMab 10F8 inhibited the binding of labeledIL-8 to neutrophils in a dose-dependent fashion. The murine anti-IL-8antibody also was able to inhibit binding of labeled IL-8 to neutrophilsin a dose-dependent fashion, but 10F8 was consistently more potent thanthe murine antibody in inhibiting IL-8 binding to neutrophils. In theexperiment shown in FIG. 7B, the IC₅₀s of 10F8 (hybridoma-derived) (10F8H) and 10F8 (transfectoma-derived) (10F8 T) were 0.19 nM and 0.30 nM,respectively.

Overall, the foregoing results demonstrate that HuMab 10F8 inhibits thebinding of IL-8 to its receptors in a dose-dependent fashion, and isable to inhibit this binding at lower concentrations than a commerciallyavailable murine anti-IL-8 antibody.

Example 7 Inhibition of IL-8 Mediated Neutrophil Chemotaxis

The ability of HuMab 10F8 and a murine anti-IL-8 antibody (MAb 6217.111,R & D Systems) to inhibit IL-8 induced neutrophil migration wasevaluated utilizing a chemotaxis assay.

Neutrophils were incubated in one chamber of a transwell plate. IL-8(rhIL-8) was incubated with varying concentrations of HuMab 10F8, themurine anti-IL-8 MAb, and a control antibody in the other chamber of thetranswell plate. The assay was incubated at 37° C. for two hours, andcell migration was quantified.

The data depicted in FIG. 8A show that neutrophil chemotaxis wasinhibited in a dose-dependent manner by HuMab 10F8 and also by themurine IL-8-specific antibody. The control antibody did not inhibitchemotaxis. These data demonstrate that HuMab 10F8 can inhibitneutrophil migration, an important function of IL-8 in vivo.

The ability of HuMab 10F8 to inhibit transmigration of human neutrophilstowards human IL-8 was furthermore studied utilizing a Boyden chamber.

Human IL-8 (10⁻⁸M) was incubated with varying concentrations of HuMab10F8 in the lower compartment of the Boyden chamber. Neutrophils (4×10⁵cells) were incubated in the upper compartment. The assay was incubatedat 37° C. for 1 hour, and cell migration was quantified.

The data depicted in FIG. 8B show that neutrophil chemotaxis wasinhibited in a dose-dependent manner by HuMab 10F8. These data alsodemonstrate that HuMab 10F8 can inhibit neutrophil migration, animportant function of IL-8 in vivo.

Example 8 Change of IL-8 Induced Adhesion Molecule Expression onNeutrophils

Human neutrophils show increased expression of CD11b (Mac-1) anddecreased expression of L-selectin (CD62L) when stimulated with IL-8.The capacity of 10F8 to inhibit the IL-8 induced changes in expressionof adhesion molecules on PMN was studied by flow cytometry. Briefly, 100μl of 1:5 diluted whole blood was incubated with serial dilutions of10F8 (5, 2.5, 1.25, 0.625, 0.312, 0 μg/mL) in the absence or presence of25 ng/mL recombinant human IL-8 (rh-IL-8, 72 aa, Peprotech) in 96-wellflat bottomed culture plates, to a final volume of 200 μl per well, for2 hours at 37° C. and 5% CO₂. Thereafter, cells were spun down and thesupernatant was stored at −20° C. until further assessment for thepresence of lactoferrin. Cells were replenished with 200 μl cold FACSbuffer (PBS, supplemented with 0.02% (v/v) azide and 0.1% (w/v) BSA) andwashed twice with 200 μl cold FACS buffer. Cells were spun down andincubated with 1.5 mg/mL PE-conjugated mouse anti-human CD11b (BectonDickinson) and 1.5 mg/mL FITC-conjugated mouse anti-human CD62L (BectonDickinson) to a final volume of 20 μl per well, for 30 minutes at 4° C.in the dark. Thereafter, erythrocytes were lysed with FACSlysis bufferaccording to the manufacturers protocol (FACSlysing solution kit, BectonDickinson, cat#349202). Subsequently, cells were washed with cold FACSbuffer. Fluorescence intensity of cells was analyzed by flow cytometry(FACS Calibur, Becton Dickinson) using Cell Quest software.

Stimulation of PMN with IL-8 resulted in an altered expression ofadhesion molecules, e.g., an enhanced expression of CD11b and adecreased expression of CD62L due to shedding of the molecule. The meanbaseline expression of CD11b on PMN was 1538±37 (n=3) MFI (MeanFluorescence Intensity, a measurement for the number of molecules percell) and the expression of CD11b was up-regulated to 2341±274 unitsafter stimulation of cells with 25 ng/mL IL-8. Baseline expression ofCD62L was 274±24 (n=3) MFI and the expression of CD62L was downregulated to 176±60 units after addition of 25 ng/mL IL-8. In thepresent study, it was demonstrated that 10F8 is capable of inhibitingthese IL-8 induced changes in expression of cell surface molecules. Thecell surface expression of CD11b, as well as CD62L, was measured by flowcytometry after stimulation of cells with 25 ng/mL IL-8 alone or in thepresence of 10F8 or an irrelevant control antibody. FIG. 9A shows theexpression of CD62L on IL-8 stimulated PMN. In the presence of anirrelevant isotype control antibody (asterisks), the MFI on thesestimulated PMN fluctuated around 150, indicating that this antibody doesnot block IL-8 mediated PMN activation. However, when cells werestimulated with IL-8 in the presence of increasing concentrations of10F8 (closed squares), reduced IL-8 mediated CD62L shedding wasobserved, in a dose-dependent way indicative for an inhibition of IL-8induced cell activation by 10F8. Consistent with this, 10F8 (closedsquares) reduced IL-8-mediated CD11b up-regulation, whereas anirrelevant isotype control antibody (asterisks) failed to show aneffect, i.e., the expression level of CD11b remained unaffected (MFIaround 2400) (n=3; FIG. 9B). Increasing concentrations of 10F8 werecorrelated with lesser expression of CD11b, clearly demonstrating aninhibitory effect of 10F8 on PMN activation by IL-8.

In summary, the results shown in FIGS. 9A and 9B demonstrate that HuMab10F8 is capable of inhibiting: a) the increased expression of CD11b onneutrophils that is mediated by IL-8; b) the shedding of L-selectin(CD62L) on the surface of neutrophils that is mediated by IL-8.

Example 9 IL-8 Present in Pustulosis Palmoplantaris (PPP) Material

Sterile material was obtained from blisters of PPP patients (pppblister), eczema patients or healthy volunteers. In addition patientsand healthy controls provided test material by wrapping the feet orhands in foil containing 2 mL phosphate buffered saline (PBS). Thewrapped feet or hands were placed in a 37° C. water bath to facilitatediffusion of blister content into the PBS (ppp water bath). Blistermaterials or water bath materials were analysed for the presence of CXCchemokines or complement factor C5a by commercial available ELISAs(IL-8: Pelikine Compact™, CLB, Amsterdam, The Netherlands; or IL-8:Quantikine® Human IL-8 kit, R&D kit; GRO-α: Quantikine® Human GRO-α kit,R&D Systems; ENA-78: Quantikine® ENA-78 kit, R&D Systems; C5a kit,Opteia, BD-Pharmingen). Normal human serum was used as negative control.ELISAs were performed according to the manufacturer's protocols.

The data depicted in FIG. 10 show that IL-8 is present in patientmaterial obtained from blisters and the water bath material, whereasIL-8 could not be demonstrated in normal serum from a healthy volunteer.IL-8 was measured on two separate occasions and it was demonstrated thatthe IL-8 content in the patient material did not decrease in time. GRO-αwas also present in the patient materials, albeit to a lesser extent.The CXC chemokine ENA-78 could not be demonstrated in any of theobtained materials.

Table 3 shows that IL-8 is present in PPP patients, whereas IL-8 wasundetectable in water bath material obtained from an eczema patient orhealthy control. GRO-α was also present in PPP patient material, albeitto a lesser extent. The samples were measured on 3 separate occasionsand demonstrated that the IL-8 or GRO-α content in the patient materialdid not decrease in time and was not degraded by presence of proteases.

TABLE 3 IL-8 GRO-a Sample No. μg/ml SEM n μg/ml SEM n No. 1 - Fluid frompustules (ppp) 17 3 3 2 0 1 No. 2 - Fluid from pustules (ppp) 517 213 339 8 2 No. 3 - Foot wash fluid (ppp) 52 2 3 21 4 2 No. 4 - Foot washfluid (ppp) 15 3 3 42 6 2 No. 5 - Foot wah fluid (ppp) 15 4 3 45 1 2 No.6 - Hand wash fluid (eczema) 0 0 3 0 0 2 No. 7 - Hand wash fluid(control) 0 0 3 0 0 2 No. 8 - Fluid from pustules (ppp) 32 16 2 2 0 1No. 9 - Foot wash fluid (ppp) 45 13 2 32 13 3

FIG. 11 shows the results of the measurement of chemokines present infeet water fluid obtained from healthy controls (n=6), eczema patients(n=6) or PPP patients (n=6). Analysis was performed on log transformeddata by one-way ANOVA with Tukey test. P<0.05 was consideredsignificantly different.

Both eczema and PPP patients show significant increase of IL-8 and GRO-αas compared to the healthy controls. Comparison of the eczema and PPPpatients shows that the amount of IL-8 is significantly differentbetween eczema and PPP patients (P<0.05), whereas the amount of GRO-α isnot significantly different between the two groups (P>0.05).Furthermore, the amount of C5a was measured and although some samplesshowed an increased amount of C5a (present in both PPP and eczemagroups), no significant differences with the control group could beobserved.

These data underline the presence of IL-8 in material obtained from PPPpatients and provide a rationale for using an anti-IL-8 antibody as atherapeutic agent in this disease.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. Any combination ofthe embodiments disclosed in the dependent claims are also contemplatedto be within the scope of the invention.

INCORPORATION BY REFERENCE

All patents, pending patent applications and other publications citedherein are hereby incorporated by reference in their entirety.

We claim:
 1. An isolated recombinant monoclonal antibody which binds tohuman IL-8 comprising a variable heavy chain amino acid sequence as setforth in SEQ ID NO:12 and a variable light chain amino acid sequence asset forth in SEQ ID NO:8.
 2. An isolated recombinant monoclonal antibodywhich binds to human IL-8, wherein the antibody comprises a human heavychain variable region comprising CDR1, CDR2, and CDR3 sequencescomprising the amino acid sequences set forth in SEQ ID NOs:22, 23, and24, respectively, and a human light chain variable region comprisingCDR1, CDR2, and CDR3 sequences comprising the amino acid sequences setforth in SEQ ID NOs:16, 17 and 18, respectively.
 3. The antibody ofclaim 2, wherein the antibody is an IgG1 antibody.
 4. The antibody ofclaim 3, wherein the antibody is an IgG1κ or IgG1λ, isotype.
 5. Theantibody of claim 3, comprising an IgG1 or IgG3 heavy chain.
 6. Theantibody of claim 2, wherein the antibody has one or more of thefollowing characteristics: (i) inhibits IL-8 binding to its receptors(CXCR1 and CXCR2); (ii) inhibits IL-8 induced proinflanimatory effects;(iii) inhibits IL-8 induced chemotactic activity for neutrophils; (iv)inhibits IL-8 induced calcium flux; (v) inhibits IL-8 induced changes inexpression levels of adhesion molecules on neutrophils; (vi) inhibitsIL-8 induced increased expression of CD11b (Mac-1) and inhibits IL-8induced decreased expression of L-selectin on neutrophils; or (vii) doesnot cross-react with related chemokines selected from the groupconsisting of human GRO-alpha, human GRO-beta, human IP-10 and humanNAP-2.
 7. The antibody of claim 2 having a dissociation equilibriumconstant (KD) of approximately 10⁻⁸ M or less, when determined bysurface plasmon resonance (SPR) technology using recombinant human IL-8as the analyte and the antibody as the ligand.
 8. The antibody of claim2 which is an antibody fragment or a single chain antibody.
 9. Theantibody of claim 2 which is a binding-domain immunoglobulin fusionprotein comprising (i) a variable heavy chain amino acid sequence as setforth in SEQ ID NO:12, fused to a variable light chain amino acidsequence as set forth in SEQ ID NO:8 via a linker peptide, that is fusedto an immunoglobulin hinge region polypeptide, (ii) an immunoglobulinheavy chain CH2 constant region fused to the hinge region, and (iii) animmunoglobulin heavy chain CH3 constant region fused to the CH2 constantregion.
 10. The antibody of claim 2, produced by a hybridoma whichincludes a B cell obtained from a transgenic non-human animal, in whichV-(D)-J gene segment rearrangements have resulted in the formation of afunctional human heavy chain transgene and a functional human lightchain transgene, fused to an immortalized cell.
 11. A bispecific ormultispecific molecule comprising the antibody according to claim 2, anda binding specificity for a human effector cell.
 12. The molecule ofclaim 11, wherein the binding specificity for a human effector cell isselected from the group consisting of a binding specificity for a humanFc gamma receptor, a human Fc alpha receptor, and a T cell receptor. 13.A composition comprising the antibody of claim 2 and a pharmaceuticallyacceptable carrier.
 14. The composition of claim 13, comprising at leastone therapeutic agent.
 15. The composition of claim 14, wherein theagent is selected from the group consisting of agents for treatinginflammatory or hyperproliferative skin disorders, and anti-inflammatoryagents.